禽传染性支气管炎病毒基因双顺反子DNA疫苗及mRNA3的分子特性研究
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摘要
1、本研究采用PCR扩增获取鸡的IL-2、IL-18基因,将其插入pMD18-T载体中进行测序鉴定。对含有S1、M、N目的基因的pIBVS1、pIBVM、pIBVN质粒进行测序分析。结果表明,IL-2、IL-18、S1、M、N基因全长分别为432bp、536bp、1671bp、678bp和1230bp,鉴定的目的基因正确。以pDsRed-Express-N1载体为骨架,以内部核糖体进入位点(internal ribosome entry site)序列为连接元件构建绿色和红色荧光基因的双顺反子表达质粒pIRES-EGFP/Red,转染Vero细胞后观察到绿色和红色荧光基因均获得表达。构建的pIRES-EGFP/Red载体含有卡那霉素抗性基因,比含有氨苄抗性的双顺反子载体具有更高的生物安全性,为今后开展各种病原微生物双顺反子DNA疫苗的研制提供研究工具。
2、用S1、M、N基因与IL-2、IL-18基因分别替换pIRES-EGFP/Red质粒中的荧光基因,构建双顺反子表达质粒pIRES-S1/IL2、pIPES-M/IL2、pIRES-N/IL2、pIRES-S1/IL18、pIPES-M/IL18和pIRES-N/IL18及其单基因表达质粒,转染Vero细胞,利用RT-PCR及间接免疫荧光检测质粒在体外的表达。RT-PCR检测表明转染了重组质粒的Vero细胞中均能特异的扩增出S1、M、N和(或)IL-2、IL-18基因片段;间接免疫荧光检测表明,转染了重组质粒的细胞显示出黄绿色荧光,而转染了空质粒的细胞则没有显示出任何特异荧光。本研究为IBV结构基因和细胞因子双顺反子DNA疫苗免疫试验提供了材料。
3、采用本课题组获得专利(专利号为200410081443.4)的质粒提取、纯化方法制备质粒,将质粒溶液(1mg/mL)与脂质体溶液(10mg/mL)等体积混合配制成DNA疫苗。将配制的DNA疫苗通过腿部肌肉多点注射7日龄非免疫雏鸡,28日龄加强免疫一次。分别在免疫前和免疫后7、14、21、28、35d采血检测ELISA抗体效价和外周血CD3+、CD4+、CD8+T淋巴细胞亚群的数量。二免后两周用IBV强毒进行攻毒。ELISA抗体水平结果显示:pIRES-S1/IL2、pIPES-M/IL2、pIRES-N/IL2疫苗组产生的抗体水平高于pIRES-S1、pIRES-M、pIRES-N疫苗组,在免疫后14-35d,差异显著(P＜0.05);pIRES-M/IL18、pIPES-N/IL18疫苗组产生的抗体水平也高于pIpES-M、pIRES-N疫苗组,在免疫后14-35d,差异显著(P＜0.05),pIRES-S1/IL2、pIRES-S1/IL18疫苗组产生的抗体水平与pIRES-S1+pIRES-IL2、pIRES-S1+pIRES-IL18疫苗组相比在免疫后21-35d差异显著(P＜0.05):外周血CD3+、CD4+、CD8+T淋巴细胞亚群数量检测结果显示:pIRES-S1/IL2、pIRES-M/IL2、pIRES-N/IL2疫苗组在免疫后7-28d,与pIRES-S1、pIRES-M、pIRES-N疫苗组相比差异显著(P＜0.05),pIRES-S1/IL18、pIRES-M/IL18、pIRES-N/IL18疫苗组在免疫后14-28d CD3+、CD4+T、CD8+T淋巴细胞亚群的数量高于pIRES-S1、pIRES-M、pIRES-N疫苗组,差异显著(P＜0.05)。pIRES-S1/IL2、pIRES-S1/IL18疫苗组的CD3+、CD4+、CD8+T淋巴细胞亚群数量较pIRES-S1+pIRES-IL2、pIRES-S1+pIRES-IL18 DNA疫苗组高,但差异不显著(P＞0.05)。攻毒结果表明,双顺反子DNA疫苗组的保护率介于65%～85%,其中RES-S1/IL2、pIRES-S1/IL18疫苗组的保护率分别为85%和75%,高于混合质粒pIRES-S1+pIRES-IL2、pIRES-S1+pIRES-IL18 DNA疫苗组的70%和60%。其中pIRES-S1/IL2疫苗组的保护率85%高于灭活疫苗组的攻毒保护率75%。本研究研制出的IBV结构蛋白与IL-2、IL-18双顺反子DNA疫苗,为提高IBV DNA疫苗的免疫效率提供了新的途径。
4、采用RT-PCR获取IBV E基因,用以替换pIRES-M/Red质粒中的红色荧光基因,构建双顺反子表达质粒pIRES-M/E,转染Vero细胞后进行表达检测。采用本论文第三章的方法制备DNA疫苗和进行动物免疫试验。结果表明:在免疫后7-35d,pIRES-M/E免疫组产生的抗体水平及外周血T淋巴细胞亚群数量高于pIRES-M组,但差异不显著(P＞0.05);pIRES-M/E、pIRES-M组对强毒的攻击保护率分别为55%和40%,且低于灭活疫苗的攻毒保护率(75%)。本研究构建了IBV的M基因和E基因双顺反子DNA疫苗并对其免疫原性进行了研究,结果初步认为E基因对M基因免疫的协同作用不明显。
5、采用RT-PCR方法对IBV mRNA3(3a3b3c)区域进行了克隆、测序及分子特性分析,测序表明3a3b3c片段大小为734bp,包含完整的3a、3b、3c(E)基因,与GenBank上公布的58株IBV毒株的相应序列的同源率介于78.2%～96.1%之间,与CK/CH/LGD/04II毒株和CK/CH/LSC/99I毒株的同源率最高(96.1%和95.9%)。对3a3b序列的RNA二级结构进行预测,结果表明两者具有相同的二级结构,但二级结构的稳定性存在差异。进一步构建3a3b的双荧光基因共表达质粒p3a3b-EGFP/Red,并对其IRES功能进行检测,结果没能检测到3a3b序列下游红色荧光基因的表达。本研究表明3a3b3c序列的分子特征与毒株的遗传变异存在相关性,为IBV的分子流行病学研究提供了新的途径。3a3b基因片段的内部核糖体进入位点功能要弱于脑心肌炎病毒的IRES序列,因此筛选高效的IRES序列用于新疫苗载体的构建还需要进一步研究。
1.Chicken interleukin-2 and interleukin-18 gene was amplified by polymerase chain reaction (PCR)from pDNAIL2 and pDNAIL18 plasmids,then ligated into pMD18-T vector.The pMD18-IL2,pMD18-IL18,pIBVS1,pIBVM and pIBVN recombinant plasmids which contain the IL-2,IL-18,S1,M and N gene were sequenced.The sequence analysis results showed that the open reading frame of IL-2,IL-18,S1,M and N gene was 432bp,536bp,1671bp,678bp and 1230bp,respectively.The correctness of target gene provides a reliable material for further experiments.In this study,we have constructed a bicistronic plasmid pIRES-EGFP/Red by using pDsRed-Express-N1 vector for the skeleton.This plamid contained two fluorescent protein genes which were inserted into the multiple cloning sites(MCS)located on either side of the internal ribosome entry site.Then the pIRES-EGFP/Red was transfected into Vero cells,green fluorescence and red fluorescence could be observed in transfected cells by using fluorescence microscope,indicating that the modified expression vector could correctly express two heterologous proteins at the same time.It is the first time to construct a bicistronic expression vector pIRES-EGFP/Red which introduced kanamycin resistance as a selection marker.Compared to the commercialization vector pIRES,it has a higher bio-safety.It provides a new research tool for the development of bivalent DNA vaccines of various pathogenic microorganism.
2.The S1,M,N gene of avian infectious bronchitis virus and interleukin-2,interleukin-18 gene were inserted into the pIRES-EGFP/Red plasmid,respectively,and generated the bicistronic plasmids pIRES-S1/IL2,pIRES-M/IL2,pIRES-N/IL2,pIRES-S1/IL18,pIRES-M/IL18 and pIRES-N/IL18.Identification of the bicistronic plasmids was performed by restriction enzyme digestion and DNA sequencing.The plasmids were transfected into Vero cells by lipofectamine, and the expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay.The results of restriction enzyme digestion and DNA sequencing showed that the bicistronic expression plasmids were constructed successfully.The mRNA transcription of S1,M,N gene which were in the upstream of IRES and IL-2,IL-18 gene which were in the downstream of IRES,could be detected by RT-PCR in the Vero cells which were transfected with plasmids.The expression of S1,M and N protein could be detected by indirect immunofluorescent assay in the Vero cells which were transfected with plasmids.In my knowledge there was no reports about the construction of co-expression vectors of IBV S1,M,N andIL-2,IL-18.before in the world.This study might provide materials for the further development of DNA vaccine against IBV.
3.In this study,the IRES-S1/IL2,pIRES-M/IL2,pIRES-N/IL2,pIRES-S1/IL18, pIRES-M/IL18,pIRES-N/IL18,pIRES-S1,pIRES-M,pIRES-N,pIRES-IL2 and pIRES-IL18 plasmids used for vaccination were extracted from JM109,and purified by a Chinese invention patent method which was established previously in our lab.The diluted plasmids(1mg/mL)were encapsulated by liposome(10mg/mL)in equal volume,and administered to the 7-day-old chickens by intramuscularly injection.After three weeks later,the chickens were boosted by DNA vaccine.Two weeks after boosting,chickens were challenged by virulent IBV strain.Fluorescence activated cell sorter and indirect ELISA methods were used to detect the number of CD3+, CD4+and CDS+ T lymphoctyes and anti-IBV antibody.The results of specific antibody show that there was a significant difference(P＜0.05)in ELISA antibody levels elicited by pIRES-S1/IL2, pIRES-M/IL2,pIRES-N/IL2 than that of pIRES-S1,pIRES-M,pIRES-N DNA vacccines since the 14th-day after first inoculation.There was a significant difference(P＜0.05)in ELISA antibody levels elicited by pIRES-M/IL18,pIRES-N/IL18 than that of pIRES-M,pIRES-N DNA vacccines since the 14th-day after first inoculation.ELISA antibody levels elicited by pIRES-S1/IL2, pIRES-S1/IL18 was higher than that of pIRES-S1+pIRES-IL2,pIRES-S1+pIRES-IL18,but there was no significant difference(P＞0.05).The percentage of CD3+,CD4+,CD8+ T-lymphocytes from chickens immunized with pIRES-S1/IL2,pIRES-M/IL2,pIRES-N/IL2 was significantly higher(P＜0.05)than pIRES-S1,pIRES-M,pIRES-N at the 7-28d after the first vaccination.The percentage of CD3+,CD4+,CD8+ T-lymphocytes significantly higher(P＜0.05)was observed from chickens immunized with pIRES-S1/IL18,pIRES-M/IL18,pIRES-N/IL18 than pIRES-S1, pIRES-M,pIRES-N at the 14-28d after the first vaccination.The percentage of CD3+,CD4+, CD8+ T-lymphocytes was higher in the pIRES-S1/IL2,pIRES-S1/IL18 than in the pIRES-S1+pIRES-IL2,pIRES-S1+pIRES-IL18 at the 14-28d after the first vaccination,but there was no significant difference(P＞0.05).The protection rate of bicistronic vaccines was between 65%and 85%.The protection rate of pIRES-S1/IL2,pIRES-S1/IL18 vaccine groups which was 85%,75%,was higher than pIRES-S1+pIRES-IL2,pIRES-S1+pIRES-IL18 vaccine groups which were 70%,60%.The studies on immunogenicity of IBV bicistronic DNA vaccines co-expression IBV S1,M,N and IL-2,IL-18 had no reports at home and abroad.It can provide a new way to enhance the IBV DNA vaccine immunogenicity and reduce costs.
4.IBV E gene was amplified by reverse transcription-polymerase chain reaction(RT-PCR). The PCR product of E gene was digested with restriction endonuclease and ligated into similarly digested pIRES-M/Red plasmid,then generated the bicistronic plasmid pIRES-M/E.Identification of the recombinant plasmid pIRES-M/E was performed by double enzymes digestion and DNA sequencing.The plasmid was transfected into Vero cells by lipofectamine,the mRNA transcripts of M and E gene were detected by RT-PCR.Restriction enzyme digestion and DNA sequencing showed that the bicistronic expression plasmid was constructed successfully,and the mRNA transcripts of M and E gene could be detected by RT-PCR in the Veto cells after transferred with pIRES-M/E plasmid,pIRES-M/E plasmid used for vaccination was extracted from JM109,and purified by a Chinese invention patent method which was established previously in our lab.The diluted plasmids(1mg/mL)were encapsulated by liposome(10mg/mL)in equal volume,and administered to the 7-day-old chickens by intramuscularly injection.After three weeks later,the chickens were boosted by DNA vaccine.Two weeks after boosting,chickens were challenged by virulent IBV strain.Fluorescence activated cell sorter and indirect ELISA methods were used to detect the number of CD3+,CD4+and CD8+ T lymphoctyes and anti-IBV antibody.The results of specific antibody and the percentage of CD3+,CD4+,CD8+ T lymphocytes in the pIRES-M/E group was higher than in the pIRES-M,but there was no significant difference(P＞0.05).The studies on immunogenicity of IBV bicistronic DNA vaccine co-expression IBV M and E gene had no reports at home and abroad.So it is the first time to explore the synergetic immune response of IBV M and E gene by co-expression.
5.The 3a3b3c gene was amplified by RT-PCR from avian infectious bronchitis virus,and cloned into the pMD18-T vector.Then the molecular characters of 3a3b3c DNA fragments were analyzed after sequencing.The prediction of RNA secondary structure of 3a3b sequence was carried out,and compared with the RNA secondary structure of encephalomyocarditis virus internal ribosome entry site.Further the bicistronic p3a3b-EGFP/Red was constructed for test the function of internal ribosome entry site of 3a3b sequence.The sequence analysis results showed that the length of 3a3b3c DNA fragment was 734bp.3a3b3c fragment which include three complete ORF of 3a,3b,3c(E)gene was 170bp,190bp,429bp in length.When compared with the corresponding sequence of 58 IBV strains published on the GenBank,the homologous analysis of 3a3b3c nucleotide sequence showed that the homology rate is between 78.2%and 96.1%.SAIBk strains has the highest rates of identity with CK/CH/LGD/04II and CK/CH/LSC/99I strains(96.1%and 95.9%),and has the lowest rate of identity with AJ310640 (78.2%).Phylogenetic analysis showed that SAIBk strain has the recent relationship with CK/CH/LGD/04II and CK/CH/LSC/99I strains which were isolated in China.This study indicated that the molecular characteristics of 3a363c sequence can be used to study the genetic evolution of IBV.The results of RNA secondary structure analysis showed that the 3a3b has the same stem-loop structure as encephalomyocarditis virus IRES sequence.Free energy values of RNA secondary structure of 3a3b and encephalomyocarditis virus IRES sequences was -45.5 kkal/mol and -129.6 kkal/mol,respectively,indicate that encephalomyocarditis virus IRES has more stable secondary structure than 3a3b's.After transfer the p3a3b-EGFP/Red plasmid to the Vero cells,the green fluorescent protein could be detected but not the red fluorescent protein,it showed that the internal ribosomal functions of 3a3b is too weak to detect by fluorescent gene expression methods. This is the first time for us to evaluate the internal ribosome entry site function of 3a3b,it can be used to study the function of IBV genome and screening a new IRES sequence for the development of vaccine vector.
2、用S1、M、N基因与IL-2、IL-18基因分别替换pIRES-EGFP/Red质粒中的荧光基因,构建双顺反子表达质粒pIRES-S1/IL2、pIPES-M/IL2、pIRES-N/IL2、pIRES-S1/IL18、pIPES-M/IL18和pIRES-N/IL18及其单基因表达质粒,转染Vero细胞,利用RT-PCR及间接免疫荧光检测质粒在体外的表达。RT-PCR检测表明转染了重组质粒的Vero细胞中均能特异的扩增出S1、M、N和(或)IL-2、IL-18基因片段;间接免疫荧光检测表明,转染了重组质粒的细胞显示出黄绿色荧光,而转染了空质粒的细胞则没有显示出任何特异荧光。本研究为IBV结构基因和细胞因子双顺反子DNA疫苗免疫试验提供了材料。
3、采用本课题组获得专利(专利号为200410081443.4)的质粒提取、纯化方法制备质粒,将质粒溶液(1mg/mL)与脂质体溶液(10mg/mL)等体积混合配制成DNA疫苗。将配制的DNA疫苗通过腿部肌肉多点注射7日龄非免疫雏鸡,28日龄加强免疫一次。分别在免疫前和免疫后7、14、21、28、35d采血检测ELISA抗体效价和外周血CD3+、CD4+、CD8+T淋巴细胞亚群的数量。二免后两周用IBV强毒进行攻毒。ELISA抗体水平结果显示:pIRES-S1/IL2、pIPES-M/IL2、pIRES-N/IL2疫苗组产生的抗体水平高于pIRES-S1、pIRES-M、pIRES-N疫苗组,在免疫后14-35d,差异显著(P＜0.05);pIRES-M/IL18、pIPES-N/IL18疫苗组产生的抗体水平也高于pIpES-M、pIRES-N疫苗组,在免疫后14-35d,差异显著(P＜0.05),pIRES-S1/IL2、pIRES-S1/IL18疫苗组产生的抗体水平与pIRES-S1+pIRES-IL2、pIRES-S1+pIRES-IL18疫苗组相比在免疫后21-35d差异显著(P＜0.05):外周血CD3+、CD4+、CD8+T淋巴细胞亚群数量检测结果显示:pIRES-S1/IL2、pIRES-M/IL2、pIRES-N/IL2疫苗组在免疫后7-28d,与pIRES-S1、pIRES-M、pIRES-N疫苗组相比差异显著(P＜0.05),pIRES-S1/IL18、pIRES-M/IL18、pIRES-N/IL18疫苗组在免疫后14-28d CD3+、CD4+T、CD8+T淋巴细胞亚群的数量高于pIRES-S1、pIRES-M、pIRES-N疫苗组,差异显著(P＜0.05)。pIRES-S1/IL2、pIRES-S1/IL18疫苗组的CD3+、CD4+、CD8+T淋巴细胞亚群数量较pIRES-S1+pIRES-IL2、pIRES-S1+pIRES-IL18 DNA疫苗组高,但差异不显著(P＞0.05)。攻毒结果表明,双顺反子DNA疫苗组的保护率介于65%～85%,其中RES-S1/IL2、pIRES-S1/IL18疫苗组的保护率分别为85%和75%,高于混合质粒pIRES-S1+pIRES-IL2、pIRES-S1+pIRES-IL18 DNA疫苗组的70%和60%。其中pIRES-S1/IL2疫苗组的保护率85%高于灭活疫苗组的攻毒保护率75%。本研究研制出的IBV结构蛋白与IL-2、IL-18双顺反子DNA疫苗,为提高IBV DNA疫苗的免疫效率提供了新的途径。
4、采用RT-PCR获取IBV E基因,用以替换pIRES-M/Red质粒中的红色荧光基因,构建双顺反子表达质粒pIRES-M/E,转染Vero细胞后进行表达检测。采用本论文第三章的方法制备DNA疫苗和进行动物免疫试验。结果表明:在免疫后7-35d,pIRES-M/E免疫组产生的抗体水平及外周血T淋巴细胞亚群数量高于pIRES-M组,但差异不显著(P＞0.05);pIRES-M/E、pIRES-M组对强毒的攻击保护率分别为55%和40%,且低于灭活疫苗的攻毒保护率(75%)。本研究构建了IBV的M基因和E基因双顺反子DNA疫苗并对其免疫原性进行了研究,结果初步认为E基因对M基因免疫的协同作用不明显。
5、采用RT-PCR方法对IBV mRNA3(3a3b3c)区域进行了克隆、测序及分子特性分析,测序表明3a3b3c片段大小为734bp,包含完整的3a、3b、3c(E)基因,与GenBank上公布的58株IBV毒株的相应序列的同源率介于78.2%～96.1%之间,与CK/CH/LGD/04II毒株和CK/CH/LSC/99I毒株的同源率最高(96.1%和95.9%)。对3a3b序列的RNA二级结构进行预测,结果表明两者具有相同的二级结构,但二级结构的稳定性存在差异。进一步构建3a3b的双荧光基因共表达质粒p3a3b-EGFP/Red,并对其IRES功能进行检测,结果没能检测到3a3b序列下游红色荧光基因的表达。本研究表明3a3b3c序列的分子特征与毒株的遗传变异存在相关性,为IBV的分子流行病学研究提供了新的途径。3a3b基因片段的内部核糖体进入位点功能要弱于脑心肌炎病毒的IRES序列,因此筛选高效的IRES序列用于新疫苗载体的构建还需要进一步研究。
1.Chicken interleukin-2 and interleukin-18 gene was amplified by polymerase chain reaction (PCR)from pDNAIL2 and pDNAIL18 plasmids,then ligated into pMD18-T vector.The pMD18-IL2,pMD18-IL18,pIBVS1,pIBVM and pIBVN recombinant plasmids which contain the IL-2,IL-18,S1,M and N gene were sequenced.The sequence analysis results showed that the open reading frame of IL-2,IL-18,S1,M and N gene was 432bp,536bp,1671bp,678bp and 1230bp,respectively.The correctness of target gene provides a reliable material for further experiments.In this study,we have constructed a bicistronic plasmid pIRES-EGFP/Red by using pDsRed-Express-N1 vector for the skeleton.This plamid contained two fluorescent protein genes which were inserted into the multiple cloning sites(MCS)located on either side of the internal ribosome entry site.Then the pIRES-EGFP/Red was transfected into Vero cells,green fluorescence and red fluorescence could be observed in transfected cells by using fluorescence microscope,indicating that the modified expression vector could correctly express two heterologous proteins at the same time.It is the first time to construct a bicistronic expression vector pIRES-EGFP/Red which introduced kanamycin resistance as a selection marker.Compared to the commercialization vector pIRES,it has a higher bio-safety.It provides a new research tool for the development of bivalent DNA vaccines of various pathogenic microorganism.
2.The S1,M,N gene of avian infectious bronchitis virus and interleukin-2,interleukin-18 gene were inserted into the pIRES-EGFP/Red plasmid,respectively,and generated the bicistronic plasmids pIRES-S1/IL2,pIRES-M/IL2,pIRES-N/IL2,pIRES-S1/IL18,pIRES-M/IL18 and pIRES-N/IL18.Identification of the bicistronic plasmids was performed by restriction enzyme digestion and DNA sequencing.The plasmids were transfected into Vero cells by lipofectamine, and the expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay.The results of restriction enzyme digestion and DNA sequencing showed that the bicistronic expression plasmids were constructed successfully.The mRNA transcription of S1,M,N gene which were in the upstream of IRES and IL-2,IL-18 gene which were in the downstream of IRES,could be detected by RT-PCR in the Vero cells which were transfected with plasmids.The expression of S1,M and N protein could be detected by indirect immunofluorescent assay in the Vero cells which were transfected with plasmids.In my knowledge there was no reports about the construction of co-expression vectors of IBV S1,M,N andIL-2,IL-18.before in the world.This study might provide materials for the further development of DNA vaccine against IBV.
3.In this study,the IRES-S1/IL2,pIRES-M/IL2,pIRES-N/IL2,pIRES-S1/IL18, pIRES-M/IL18,pIRES-N/IL18,pIRES-S1,pIRES-M,pIRES-N,pIRES-IL2 and pIRES-IL18 plasmids used for vaccination were extracted from JM109,and purified by a Chinese invention patent method which was established previously in our lab.The diluted plasmids(1mg/mL)were encapsulated by liposome(10mg/mL)in equal volume,and administered to the 7-day-old chickens by intramuscularly injection.After three weeks later,the chickens were boosted by DNA vaccine.Two weeks after boosting,chickens were challenged by virulent IBV strain.Fluorescence activated cell sorter and indirect ELISA methods were used to detect the number of CD3+, CD4+and CDS+ T lymphoctyes and anti-IBV antibody.The results of specific antibody show that there was a significant difference(P＜0.05)in ELISA antibody levels elicited by pIRES-S1/IL2, pIRES-M/IL2,pIRES-N/IL2 than that of pIRES-S1,pIRES-M,pIRES-N DNA vacccines since the 14th-day after first inoculation.There was a significant difference(P＜0.05)in ELISA antibody levels elicited by pIRES-M/IL18,pIRES-N/IL18 than that of pIRES-M,pIRES-N DNA vacccines since the 14th-day after first inoculation.ELISA antibody levels elicited by pIRES-S1/IL2, pIRES-S1/IL18 was higher than that of pIRES-S1+pIRES-IL2,pIRES-S1+pIRES-IL18,but there was no significant difference(P＞0.05).The percentage of CD3+,CD4+,CD8+ T-lymphocytes from chickens immunized with pIRES-S1/IL2,pIRES-M/IL2,pIRES-N/IL2 was significantly higher(P＜0.05)than pIRES-S1,pIRES-M,pIRES-N at the 7-28d after the first vaccination.The percentage of CD3+,CD4+,CD8+ T-lymphocytes significantly higher(P＜0.05)was observed from chickens immunized with pIRES-S1/IL18,pIRES-M/IL18,pIRES-N/IL18 than pIRES-S1, pIRES-M,pIRES-N at the 14-28d after the first vaccination.The percentage of CD3+,CD4+, CD8+ T-lymphocytes was higher in the pIRES-S1/IL2,pIRES-S1/IL18 than in the pIRES-S1+pIRES-IL2,pIRES-S1+pIRES-IL18 at the 14-28d after the first vaccination,but there was no significant difference(P＞0.05).The protection rate of bicistronic vaccines was between 65%and 85%.The protection rate of pIRES-S1/IL2,pIRES-S1/IL18 vaccine groups which was 85%,75%,was higher than pIRES-S1+pIRES-IL2,pIRES-S1+pIRES-IL18 vaccine groups which were 70%,60%.The studies on immunogenicity of IBV bicistronic DNA vaccines co-expression IBV S1,M,N and IL-2,IL-18 had no reports at home and abroad.It can provide a new way to enhance the IBV DNA vaccine immunogenicity and reduce costs.
4.IBV E gene was amplified by reverse transcription-polymerase chain reaction(RT-PCR). The PCR product of E gene was digested with restriction endonuclease and ligated into similarly digested pIRES-M/Red plasmid,then generated the bicistronic plasmid pIRES-M/E.Identification of the recombinant plasmid pIRES-M/E was performed by double enzymes digestion and DNA sequencing.The plasmid was transfected into Vero cells by lipofectamine,the mRNA transcripts of M and E gene were detected by RT-PCR.Restriction enzyme digestion and DNA sequencing showed that the bicistronic expression plasmid was constructed successfully,and the mRNA transcripts of M and E gene could be detected by RT-PCR in the Veto cells after transferred with pIRES-M/E plasmid,pIRES-M/E plasmid used for vaccination was extracted from JM109,and purified by a Chinese invention patent method which was established previously in our lab.The diluted plasmids(1mg/mL)were encapsulated by liposome(10mg/mL)in equal volume,and administered to the 7-day-old chickens by intramuscularly injection.After three weeks later,the chickens were boosted by DNA vaccine.Two weeks after boosting,chickens were challenged by virulent IBV strain.Fluorescence activated cell sorter and indirect ELISA methods were used to detect the number of CD3+,CD4+and CD8+ T lymphoctyes and anti-IBV antibody.The results of specific antibody and the percentage of CD3+,CD4+,CD8+ T lymphocytes in the pIRES-M/E group was higher than in the pIRES-M,but there was no significant difference(P＞0.05).The studies on immunogenicity of IBV bicistronic DNA vaccine co-expression IBV M and E gene had no reports at home and abroad.So it is the first time to explore the synergetic immune response of IBV M and E gene by co-expression.
5.The 3a3b3c gene was amplified by RT-PCR from avian infectious bronchitis virus,and cloned into the pMD18-T vector.Then the molecular characters of 3a3b3c DNA fragments were analyzed after sequencing.The prediction of RNA secondary structure of 3a3b sequence was carried out,and compared with the RNA secondary structure of encephalomyocarditis virus internal ribosome entry site.Further the bicistronic p3a3b-EGFP/Red was constructed for test the function of internal ribosome entry site of 3a3b sequence.The sequence analysis results showed that the length of 3a3b3c DNA fragment was 734bp.3a3b3c fragment which include three complete ORF of 3a,3b,3c(E)gene was 170bp,190bp,429bp in length.When compared with the corresponding sequence of 58 IBV strains published on the GenBank,the homologous analysis of 3a3b3c nucleotide sequence showed that the homology rate is between 78.2%and 96.1%.SAIBk strains has the highest rates of identity with CK/CH/LGD/04II and CK/CH/LSC/99I strains(96.1%and 95.9%),and has the lowest rate of identity with AJ310640 (78.2%).Phylogenetic analysis showed that SAIBk strain has the recent relationship with CK/CH/LGD/04II and CK/CH/LSC/99I strains which were isolated in China.This study indicated that the molecular characteristics of 3a363c sequence can be used to study the genetic evolution of IBV.The results of RNA secondary structure analysis showed that the 3a3b has the same stem-loop structure as encephalomyocarditis virus IRES sequence.Free energy values of RNA secondary structure of 3a3b and encephalomyocarditis virus IRES sequences was -45.5 kkal/mol and -129.6 kkal/mol,respectively,indicate that encephalomyocarditis virus IRES has more stable secondary structure than 3a3b's.After transfer the p3a3b-EGFP/Red plasmid to the Vero cells,the green fluorescent protein could be detected but not the red fluorescent protein,it showed that the internal ribosomal functions of 3a3b is too weak to detect by fluorescent gene expression methods. This is the first time for us to evaluate the internal ribosome entry site function of 3a3b,it can be used to study the function of IBV genome and screening a new IRES sequence for the development of vaccine vector.
引文
1.王红宁主编.禽传染性支气管炎综合防治,北京:中国农业出版社,2000.
2.苏敬良,高福译,Saif Y M主编.禽病学(第十一版),北京:中国农业出版社,2005.
3.Jia W,Karaca K,Parrish C R,et al.A novel variant of avian infectious bronchitis virus resulting from recombination among three different strains.Arch Virol,1995,140:259-271.
4.Wang H N,Wu Q Z,Huang Y,et al.Isolation and identification of infectious bronchitis virus from chickens in Sichuan,China.Avian Dis,1997,41:279-282.
5.Yu L,Jiang Y,Low S,et al.Characterization of three infectious bronchitis virus isolates from China associated with proventriculus in vaccinated chickens.Avian Dis,2001,45:416-424.
6.Liu S W,Kong X G.A new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and nonvaccinated flocks in China.Avian Pathol,2004,33:321-327.
7.Zhou J Y,Zhang D Y,Ye J X,et ah Characterization of an avian infectious bronchitis virus isolated in China from chickens with nephritis.J Vet Med B Infect Dis Vet Public Health,2004,51:147-152.
8.Bayry J,Goudar M S,Nighot P K,et al.Emergence of a nephropathogenic avian infectious bronchitis virus with a novel genotype in India.J Clin Microbiol,2005,43:916-918.
9.Alvarado I R,Villegas P,Mossos N,et al.Molecular characterization of avian infectious bronchitis virus strains isolated in Colombia during 2003.Avian Dis,2005,49:494-499.
10.Ignjatovic J,Gould G,Sapats S.Isolation of a variant infectious bronchitis virus in Australia that further illustrates diversity among emerging strains.Arch Virol,2006,151:1567-1585.
11.江国托,刘思国,康丽娟,等.鸡传染性支气管炎病毒的遗传与变异(一).养禽与禽病防治,1999,(4):6-9.
12.Cavanagh D,Davis P J,Darybyshire J H,et al.Coronavirus IBV:virus retaining spike glycopolypeptide S2 but not S1 is unable to induce virus neutralizing or haemagglutination inhibiting antibody,or induce chicken tracheal protection.Journal of General Virology,1986,67(7):1435-1442.
13.Koch G,Kant A.Nucleotide and amino acid sequence of the S1 subunit of the spike glycoprotein of the coronavirus IBV.Nuc1 Acids Res,1990,18:3063-3064.
14.Collisson E W,Pei J,Dzielawa J,et al.Cytotoxic T lymphocytes are critical in the control of infectious bronchitis yirus in poultry.Dev Comp Immunol,2000,24:187-200.
15.Seo S H,Wang L,Smith R,et al.The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection.J Virol,1997,71,7889-94.
16.Mockett A P,Cavanagh D,Brown T D.Monoclonal antibodies to the S1 spike and membrane proteins of avian infectious bronchitis coronavirus strain Massachusetts M41.J Gen Virol,1984,65:2281-2286.
17.Mockett A P.Envelope proteins of avian infectious bronchitis virus:purification and biological properties.J Virol Methods,1985,12:271-278.
18.Klumperman J,Locker J K,Meijer A,et al.Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding.J Virol,1994,68:6523-6534.
19.Schalk A K,Hawn M C.An apparently new respiratory disease of baby chicks.J Am Vet Med Assoc,1931,78:413-422.
20.Capua I,Minta Z,Karpinska E,et al.Cocirculation of four types of infectious bronchitis virus(793/B 624/I B1648 and Massachusetts).Avian Pathol,1999,28:587-592.
21.CavanaghD,Ellis M M,Cook J K A.Relationship between sequence variation in the A1spike protein of infectious bronchitis virus and the extent of cross-protection in vivo.Avian Pathol,1997,26:63-74.
22.Cavanagh D,Severe acute respiratory syndrome vaccine development:experiences of vaccination against avian infectious bronchitis coronavirus.Avian Pathol,2003,32:567-582.
23.Cook J K A,Orbell S J,Woods M A,et al.Breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous types.Avian Pathol,1999,28:471-479.
24.Darbyshire J H,Rowell J G,Cook J K A,et al.Taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures.Arch Virol,1979,61:227-238.
25.De Wit J J.Detection of infectious bronchitis virus.Avian Pathol,2000,29:71-93.
26.El Houadfi M,Jones R C,Cook J K A,et al.The isolation and characterization of six avian infectious bronchitis viruses isolated in Morocco.Avian Pathol,1986,15:93-105.
27.Farsang A,Ros C,Renstrom L HM,et al.Molecular epizootiology of infectious bronchitis Virus in Sweden indicating the involvement of a vaccine strain.Avian Pathol,2002,31:229-236.
28.Gelb J Jr,Weisman Y,Ladman B S,et al.$1 gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the United States and Israel(1996 to 2000).Avian Pathol,2005,34:194-203.
29.Hofstad M S.Cross-immunity in chickens using seven isolates of avian infectious bronchitis virus.Avian Dis,1981,25:650-654.
30.Ladman B S,Loupos A B,Gelb J Jr.Infectious bronchitis virus S1 gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization.Avian Pathol,2006,35:127-133.
31.Lambrechts C,Pensaert M,Ducatelle R,Challenge experiments to evaluate crossprotection induced at the trachea and kidney level by vaccine strains and Belgian nephropathogenic isolates of avian infectious bronchitis virus.Avian Pathol,1993,22:577-590.
32.Picault J P,Drouin P,Guittet M,et al.Isolation characterisation and preliminary cross-protection studies with a new pathogenic avian infectious bronchitis virus(strain PL-84084).Avian Pathol,1986,15:367-383.
33.廖明,辛朝安,王林川,等.鸡传染性支气管病毒S1基因在昆虫细胞中的表达.中国兽医学报.1997,6(17):539-543.
34.宋延华,刘福安.鸡传染性支气管炎病毒S工基因在昆虫细胞中的高效表达.中国兽医学报,1999,19(5):421-424.
35.戴亚斌,陈德胜,丁铲,等.表达鸡传染性支气管炎病毒SD/97/01株S1蛋白的重组杆状病毒的构 建.厦门大学学报:自然科学版,2002,41(4):487-492.
36.Song C S,Lee Y J.Induction of protective immunity in chickens vaccinated with infectious bronchitis virus Slglycoprotein expressed by a recombinant baculovirus.J Gen Virol,1998,79(4):719-723.
37.步志高,江国托,王秀荣,等.传染性支气管炎病毒S1基因DNA免疫质粒的构建及其免疫原性.中国兽医学报,1998,18(6):527-530.
38.陈洪岩,江国托,杨奇伟,等.鸡传染性支气管炎病毒S1基因免疫对鸡的保护作用.中国预防兽医学报,1999,21(4):250-252.
39.江国托,步志高,刘思国,等.鸡传染性支气管炎病毒中国流行株免疫原S1基因的分子克隆、序列分析及其DNA免疫的初步研究.生物工程学报,1999,15(3):305-309.
40.Kapcynski D R.Protection of chickens from infectious bronchitis by in ovo and intramuscular vaccination with a DNA vaccine expressing the S1 glycoprotein.Avian Dis,2003,47:272-285.
41.刘思国,康丽娟.江国托,等.鸡传染性支气管炎病毒核蛋白基因重组真核表达载体的构建.中国兽医学报,2001,21(1):14-16.
42.李晓英,王红宁,黄勇,等.禽传染性支气管炎脂质体、壳聚糖/DNA疫苗免疫效果比较研究.畜牧兽医学报,2006,37(12):1319-1322.
43.Casais R,Thiel V,Siddell S G,et al.Reverse genetics system for the avian coronavirus infectious bronchitis virus.J Virol,2001,75:12359-12369.
44.Hodgson T,Casais R,Dove B,et al.Recombinant infectious bronchitis coronavirus Beaudette with the spike protein gene of the pathogenic M41 strain remains attenuated but induces protective immunity.J Virol,2004,78:13804-13811.
45.Tomley F M,Mockett A P,Boursnell M E,et al.Expression of theinfectious bronchitis virus spike protein by recombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice.J Gen Virol,1987,68:2291-2298.
46.Yu L,Liu W,Schnitzlein W M,et al.Study of protection by recombinant fowl poxvirus expressing C-terminal nucleocapsid protein of infectious bronchitis virus against challenge.Avian Dis,2001,45:340-348.
47.Wang X,Schnitzlein W M,Tripathy D N,et al.Construction and immunogenicity studies of recombinant fowl poxvirus containing the S1 gene of Massachusetts 41 strain of infectious bronchitis virus.Avian Dis,2002,46:831-838.
48.Johnson M A,Pooley C,Ignjatovic J,et al.A recombinant fowl adenovirus expressing the S1 gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus.Vaccine,2003,21:2730-2736.
49.Zhou J Y,Cheng L Q,Zheng X J,et al.Generation of the transgenic potato expressing full-length spike protein of infectious bronchitis virus.J Biotechnol,2004,111:121-130.
50.李春,王红宁,周生,等.禽传染性支气管炎病毒S1基因玉米表达载体的构建.四川畜牧兽医,2003,30(B09):18-18.
51.Hildegund C J E.DNA疫苗.李琦涵,刘龙丁,车艳春,译.北京:化学工业出版社,2005.
52.房文亮,李泽民,揣侠,等.白细胞介素2和15基因对柯萨奇病毒VP1 DNA疫苗诱导免疫应答的影响.临床心血管病杂志,2005,21(1):33-35.
53.Min W,Lillehoj H S,et al.Adjuvant effects of IL-I beta,IL-2,IL-8,IL-15,IFN-alpha,IFN-gamma,TGF-beta4 and lymphotactin on DNA vaccinatiom against Eimeria acervulina.Vaccine,2001,20(1-2):267-274.
54.江文正.金宁一,李子健,等.IL-18和HIV-1 gag-sp120嵌合基因DNA疫苗联合免疫小鼠的免疫应答.细胞与分子免疫学杂志,2004,20(4):433-435.
55.Billaut-Mulot O,Idziorek T,Loyens M,et al.Modulation of cellular and humoral immune responses to a multiepitopic HIV-1 DNA vaccine by interleukin-18 DNA immunization/viral protein boost.Vaccine,2001,19(20-22):2803-2811.
56.Lid X J,Zhu M Z,Song G X,et al.Enhancement of herpes simplex virus-1 glycoprotein-D DNA vaccine induced specific immune responses by coimmunization with interleukin-2genetic adjuvant.Zhongguo Yi Xue Ke Xue Yuan Xue Bao,2005,27:67-72.
57.Barouch D H,Santra S,Steenbeke T D,et al.Augmentation and suppression of immune response to an HIV-1 DNA vaccine by plasmid cytokine/Ig administration.Immunol,1998,161:1875-1882.
58.程相朝,赵德明.鸡IL-18真核表达载体的构建及其对新城疫疫苗免疫增强作用的研究.畜牧兽医学报,2005,36(5):476-481.
59.Degen W G,van Zuilekom H I,Scholtes N C,et al.Potentiation of humoral immune responses to vaccine antigens by recombinant chicken IL-18(rChIL-18).Vaccine.2005,23(33):4212-4218.
60.Xiang Z Q,Spitalink K,Tran M,et al.Vaccinatiion with a plamid vetor carrying the rabies glycoprotein gene induces protective immunity against rabies virus.Virology,1994:132-140.
61.Barouch D H,Santra S,Racz P et al.Potent CD4+ T cell response elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF.Immunology,2002,168:562-568.
62.Wang J,GujarSA,Cova L,et al.Bicistronic woodchuck hepatitis virus core and gamma interferon DNA vaccine can protect from hepatitis but does not elicit sterilizing antiviral immunity.J Virol,2007,81:903-916.
63.Singh G,Parker S,Hobart P.The development of a bicistronic plasmid DNA vaccine for B-cell lymphoma.Vaccine,2002,31(20):1400-1411.
64.Mountford P S,Smith A G.Internal ribosome entry site and dicistronic RNAs in mammalian transgenesis.Trends Genet,1995,11(5):179-184.
65.Pelletier J,Sonenberg N.Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA.Nature,1988,334(6180):320-325.
66.Jang S K,Krausslich H G,Nicklin M J,et al.A segment of the 59 nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation.J Virol,1988,62(8):2636-2643.
67.詹林盛,饶林,彭剑淳,等.HCV IRES介导萤火虫荧光素酶翻译的双顺反子载体的构建与在小鼠体内的表达.中国生物化学与分子生物学报,2004.20(3):330-334.
68.Wild J,Gruner B,Metzger K,et al.Polyvalent vaccination against hepatitis B surface and core antigen using a dicistronic expression plasmid.Vaccine,1998,16:353-360.
69.郝敏麒,徐军,钟南山.尘瞒主要变应原DNA疫苗对其提取液诱导小鼠肺部变应性炎症的免疫保护作用.中华结核和呼吸杂志,2002,25:159-161.
70.Maecker H T,Hansen G,Walter D M,et al.Vaccination with allergen-IL-18 fusion DNA protects against and reverses established airway hyperreal livity in a murine asthma model.J Immunol.2001.166:959-965.
71.陈创大,余兴龙,马正海,等.细胞因子与猪瘟病毒E2基因真核表达载体的构建及其免疫增强作用.中国农业科学.2002,35(11):1408-1410.
72.Boursnell M E C,Brow T D K,Foulds I J,et al.Completion of the sequence of the genome of the Coronavirus infectious bronchitis virus.J Gen Virol,1987,68:57-77.
73.Liu D X,Cavanagh D,et al.A polycistronic mRNA specified by the coronavirus IBV.Virology,1991,184(2):531-544.
74.Jia W,Naqi S A.Sequence analysis of gene 3,gene 4 and gene 5 of avian infectious bronchitis virus strain CU-T2.Gene,1997,189(2):189-193.
75.Shen S,Wen Z L,Liu D X.Emergence of a cornavirus infectious bronchitis virus mutant with a truncated 3b gene:functional characterization of the 3b protein in pathogenesis and replication.Virology,2003,311:16-27.
76.Teri Hodgson,Poul Britton,Dave Cavanagh,et al.Neither the RNA nor the protein of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication.Virology,2006,80:296-305.
77.Liu D X,Inglis S C,etal.Internal entry of ribosomes on a tricistronic mRNA encoded by infectious bronchitis virus.Journal of Virology,1991,66(10):6143-6154.
78.Pelletier J,Sonenberg N.Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA.Nature,1988:320-334.
79.Jang S K,Krausslich H G,Nicklin M J,et al.A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosome during in vitro translation.J Virol,1988,62:2636.
80.Wang H N,Wu Q Z,nuang Y,et al.Isolation and identification of infectious bronchitis virus from chickens in Sichuan,China.Avian Dis,1997,41:279-282.
81.黄勇,王红宁,王林川,等.应用RT-PCR技术扩增禽传染性支气管炎病毒S1基因的研究.中国畜禽传染病,1998,20(5):297-298.
82.王林川,王红宁,黄勇,等.用RT-PCR和RFLP对禽传染性支气管炎病毒中国分离株的分型.病毒学报,1997,13(4):365-368.
83.余祖华,王红宁,周生,等.禽传染性支气管炎病毒S1基因毕赤酵母表达载体的构建.中国家禽,2006,28:14-16.
84.周生,王红宁,唐梦君,等.禽传染性支气管炎冠状病毒SAIBK株全基因组cDNA文库的构建与序列测定.畜牧兽医学报,2007,1:72-77.
85.Tahara H,Zitvogel L,Storkus W J,et al.Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector.J Immunol,1995,154(12):6466-6674.
86.David L,Suarez,Stacey Schultz-Cherry.The effect of eukaryotic expression vectors and adjuvants on DNA vaccines in chickens using an avian influenza model.Avian Disease,2000,44:861-868.
87.Alexander L,Lu H H,Gromeier M,Wimmer E.Dicistronic polioviruses as expression vectors for foreign genes.AIDS Res Hum Retorviruses,1994,10(2):57-60.
88.Pugachev K V,Tzeng W P,Frey T K.Development of a rubella virus vaccine expression vector:use of a picornavirus internal ribosome entry site increases stability of expression.J Virol,2000 Nov,74(22):10811-10815.
89.Benvenisti L,Rogel A,Kuznetzova L,et al.Gene gun-mediate DNA vaccination against foot-and-mouth disease virus.Vaccine,2001,19(28-29):3885-3895.
90.Osorio J E,Powell T D,Frank R S,etal.Recombinant raccoon pox vaccine portects mice against lethal plague.Vaccine,2003:21(11-12):1232-1238.
91.Lewis J C.Applications of reporter genes.Analytical Chemistry,1998,70(17):579-585.
92.薛丽香.童坦君,张宗玉,等.报告基因的选择及其研究趋向.生理科学进展,2002,33(4):364-366.
93.刘兆球,姜世金,常维山,等.传染性支气管炎病毒S1基因的真核表达及免疫原性检测.中国兽医学报,2005,25(3):241-243.
94.焦红梅,焦新安,殷月兰,等.鸡传染性支气管炎病毒S1基因的真核表达及其产物的免疫原性.扬州大学学报(农业与生命科学版),2006,27(2):44-47.
95.Ignjatovic J,Galli L.Immune responses to structural proteins of avian infectious virus.Avian Pathol,1995,24:313-332.
96.Borman A M,Mercier P L,Girard M,et al.Comparison of picornaviral IRES-driven internal initiation of translation in cultured cells of different origins.Nucleic Acids Res,1997,25(5):925-932.
97.Hiroyuki Mizuguchi,Zhili Xu,Akiko Ishii-Watabe,et al.IRES-Dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector.Molecular Ther,2000,1(4):376-382.
98.Saporito-Irwin S M,Geist R T,Gutmann D H.Ammonium acetate protocol for the preparation of plasmid DNA suitable for mammalian cell transfections.Biotechniques.1997,23(3):424-427.
99.姜世金,刘兆球,常维山,等.传染性支气管炎病毒S1基因真核表达质粒的构建及其在CEF中的转染.中国兽医杂志,2004,40(12):17-18.
100.赵宝华,马同锁,杨虹,等.鸡传染性支气管M41毒株S1基因的克隆与真核表达研究.动物医学进展,2005,26(1):71-75.
101.周生,王红宁.禽传染性支气管炎病毒分离株的全基因组及S1、M、N基因DNA疫苗研究.四川农业大学博士学位论文,2007.
102.陆彬.药物新剂型与新技术.北京:人民卫生出版社,1998,107-110.
103.Ishii N,Fukushima J,Kaneko T,et al.Cationic liposomes area strong adjuvant for a DNA vaccine of human immunodeficiency virus type.Aids Research in human retroviruses,1997,13:1421-1428.
104.彭保卫,郭祀远,杨富强,等.HBV DNA疫苗阳离子脂质体复合物转染小鼠实验研究.广东药学学院学报,2002.18(4):294-297.
105.Iqbalet M,Lin W,Jabbal Gill I,et al.Nasal delivery of chitosan-DNA plasmid expressing epitopes of respiratory syncytial virus(RSV)induces protective CTL responses in BALB/c mice.Vaccine,2003,21(13-14):1478.
106.Roy K,Mao H Q,Huang S K,et al.Oral gene delivery with chitosan-DNA nanoparticles generates immunologic protection in a murine peanut a11ergy model.Natmed,1999,5:387..
107.Cavanagh D.,Severe acute respiratory syndrome vaccine development:experiences of vaccination against avian infectious bronchitis coronavirus,Avian Pathol,2003,32:567-582.
108.De Wit J J.Detection of infectious bronchitis virus,Avian Pathol,2000,29:71-93.
109.Henke A,Rohland N,Zell R,et al.Co-expression of interleukin-2 by a bicistronic plasmid increases the efficacy of DNA immunization to prevent influenza virus infections.Intervirology,2006,49:249-52.
110.Henke A,Chiang C S,Zell R,et al.Co-expression of interleukin-2 to increase the efficacy of DNA vaccine-mediated protection in coxsackievirus B3-infected mice. Antiviral Res,2004,64:131-136.
111.Barouch D H,Santra S,Steenbeke T D et al.Augmentation and suppression of immune response to an HIV-1 DNA vaccine by plasmid cytokine/Ig administration.Immunol,1998,161:1875-1882.
112.Marshall D J,Rudnick K A,McCarthy S G,et al.Interleukin-18 enhances Th1 immunity and tumor protection of a DNA vaccine.Vaccine,2006,24:244-53.
113.Kim J J,Yang JS,Dang K,et al.Engineering enhanacement of immune responses to DNA-based vaccines in a prostate cancer model in rhesus macaques through the use of cytokine gene adjuvants.Clin Cancer Res,2001,7(Suppl 3):882-889.
114.Timms L M,Bracewell C D.Cell mediated and humoral immune response of chickens to live infectious bronchitis vaccines.Res Vet Sci,1981,31:182-189.
115.Chow Y H,Huang W L,Cli W K,et al.Improvement of hepatitis B virus DNA vaccine by plasmids coexpressing hepatitis B surface antigen and interleukin-2.J Virol,1997,71(1):169-178.
116.Li W R,Niu B,Wang J W,et al.Coexpression of interleukin-2 enhances the immunization effect of aDNAvaccine expressing herpes simplex 1 glycoprotein D:Acta Virol,2006,50:251-256.
117.Kamath A T,Hanke T,Briscoe H,et al.Co-immunization with DNA vaccines expressing granulocyte-macrophage colony-stimulating factor and mycobacterial secreted proteins enhances T-cell immunity,but not protective efficacy against Mycobacterium tuberculosis.Immunology,1999,96:511.
118.洪晓武,窦骏,陈国兵,等.mGM-CSF协同抗肿瘤作用研究进展.实用肿瘤杂志,2003,18:154.
119.Lgnjatovic J,Galli L.The S1 glycoprotein but not the N or M proteins of avian infectious bronchitis virus induces protection in vaccinated chickens.Arch Virol,1994,138:117-134.
120.Wang C H,Huang Y C.Relationship between serotypes and genotypes based on the hypervariable region of the S1 gene of infectious bronchitis virus.Arch Virol,2000,145:291-300.
121.Corse E,Machaser C E,The cytoplasmic tail of infectious bronchitis virus E protein directs Golgi targeting.J Virol,2002,76(3):1273-1284.
122.张德勇,周继勇.基因IBV重组表达蛋白的抗体检测技术及基因免疫研究.浙江大学博士学位论文,2005.
123.David F Stern,Bartholomer M Sefton.Coronavirus proteins:structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins.Virology,1982,804-812.
124.刘胜旺,贺秀瑗,孔宪刚,等.鸡传染性支气管炎病毒中国分离株M基因的分子特征.中国兽医学报,2001,23(6):404-407.
125.Ng Lisa F P,Liu D X,Identification of a 24-kDa polypeptide processed from the Coronavires infectious bronchitis virus 1a polyprotein by the 3c-like proteinase and determination of its cleavage sites.Virology,1998,243:388-395.
126.Lim Z P,Liu D X,The missing link in coronavirus assembly.Retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-Golgi compartments and physical interaction between the envelope and membrane proteins.J Biol Chem,2001,276(20):17515-17523.
127.Junko M,John F R,Akihiko M,et al.Rapid communication:Membrane topology of coronavirus E protein.Virology,2001,281(i):163-169.
128.Dave cavanagh.Coronavirus avian infectious bronchitis virus.Vet Res,2007,38:281-297.
129.黄亚东,王林川,郑青,等.鸡传染性支气管炎病毒H株纤突蛋白S1基因的克隆及其在毕赤酵母中表达.病毒学报,2003,19(2):144-148.
130.周继勇,JimmyKwang.传染性支气管炎病毒核衣壳蛋白基因变异及在大肠杆菌中的表达.浙江大学学报:农业与生命科学版,2003,29(1):1-9.
131.刘胜旺,杜恩歧,孔宪刚,等.鸡传染性支气管炎病毒核蛋白基因的体外表达和纯化.中国实验动物学杂志,2002,12(5):302-302.
132.陈萍,王红宁.间接ELISA检测IBV DNA疫苗免疫抗体及IBV M基因表达研究,四川农业大学硕士论文,2005
133.Binns M M,et al.Israel Journal of veterinary Medicine,1986,42(2):124-127.
134.Evans S,Cavanagh D.Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus.J Gem Virol,2000,81(12):2855-2865.
135.Yu L,Liu W,Schnitzlein W M,et al.Study of protection by recombinant fowl poxvirus expressing C-terminal nucleocapsid protein of infectious bronchitis virus against challenge.Avian Dis,2001,45:340-348.
136.田占成,孙永科,王云峰,等.表达鸡传染性支气管炎病毒S1基因重组鸡痘病毒对SPF鸡的免疫保护作用.畜牧兽医学报,2006,37(6):580-586.
137.焦红梅,焦新安,田华荣,等.减毒鼠伤寒沙门氏菌运送的鸡传染性支气管炎病毒S1基因DNA疫苗及其免疫效力研究.病毒学报,2007,23(3):212-117.
138.Kapcynski D R.Protection of chickens from infectious bronchitis by in ovo and intramuscular vaccination with a DNA vaccine expressing the S1 glycoprotein.Avian Dis,2003,47:272-285.
139.杨帆,王红宁,李春,等.禽传染性支气管炎病毒四川分离株M基因的克隆及真核表达载体构建。四川畜牧兽医,2003,30(B09):19-19.
140.Xin K Q,Hamajima K,Sasar S,et al.Intranasal administration of human immunodeficiency virus type 1(HIV1)DNA vaccine with interleukin2 expression plasmid enhance cell mediated immunity against HIV.Immunology,1998,94(3):438-444.
141.Kim J J,Ayyavoo V,Bagarass M L,et al.In vivo engineering of a cellular immune response by coadministration of IL-12 expression vetor with a DNA immunogen.J Immunol,1997,158:816-826.
142.Gherardi M M,Ramirez J C.Esteban M.Towards a new generation of vaccine:the cytokine IL-12 as an adjuvant to enhance cellular inmmune responses to pathogens during prime-booster vaccination regimens.Histol Histopathol,2001,16(2):655-667
143.Okada H,Giezeman-Smits K M,Tahara H,et al.Effective cytokine gene therapy against an intracranial glioma using a retrovirally transduced IL-4 plus HSV tk tumor vaccine.Gene Ther,1999,6(2):219-226.
144.Kim S H,Cho D,Hwang S Y,et al.Efficient induction of antigenspecific T helper type Ⅰ mediated immune responses by intramuscular injection with ovalbumin/interleukin-18fusion DNA.Vaccine,2001,19(30):4107-4114.
145.Sin J I,Kim J J,Boyer J D,et al.In vivo modulation of vaccine induced immune responses toword a Th1 phenotype increases potency and vaccine effectiveness in herpes simplex virus type 2 mouse model.J Virol,1999,73(1):501-509.
146.Geissler M,Gesien A,Tokushige K,et al.Enhancement of cellular and hunoral immune responses to hepatitis C titus core protein using DNA-based vaccinges augmented with cytokine-expressing plasmids.J Immunol,1997,158:1231-1237.
147.Kim J J,Yang J S,Manson K M,et al.Coimmunization with IFN-gamma or IL-2,but Not IL-13 or IL-4 cDNA Can Enhance Th1-Type DNA Vaccine-Induced.Journal of Interferon &Cytokine Research,2000,20(3):311-319.
148.Min W,Lillehoj H S,et al.Adjuvant effects of IL-1 beta,IL-2,IL-8,IL-15,IFN-alpha,IFN-gamma,TGF-beta4 and lymphotactin on DNA vaccinatiom against Eimeria acervulina.Vaccine,2001,20(1-2):267-274.
149.Lim Y S,Kang B Y,Kim E J,et al.Potentiation of antigen-specific,T hi immune response by multiple DNA vaccination with an ovalbumin/interferon-γ hybrid construct.Immunol,1998,94:135-141.
150.Mbawuike I N.Enhancement of the protecting efficacy of inactivated influenza A vaccine in aged mice by IL-2-1iposomes.Vaccine,1990,8:347-352.
151.Operschall E,SchuhT,Heinzerling L,et al.Enhanced protection against viral infection by co-administration of plasmid DNA coding for viral antigen and cytokines in mice.J Clin Virol,1999,13:17-22.
152.杜德伟,周永兴,冯志华,等.IL-12及HBV基因疫苗共同免疫小鼠的效果.世界华人消化杂志,2000,8(2):128-130.
153.Lee S W,Cho J M,Sung Y C,et al.Optimal induction of hepatitis C virusenvelope-specific immunity by bicistronic plasmid DNA inoculation with thegranulocyte-macrophage colony-stimulating factor gene.J Virol,1998,72:8430-8436.
154.Parker S E,Monteith D,.Morton H,et al.Safety of a GM-CSF adjuvant-plasid DNAmalaria vaccine.Gene Ther,2001,8(13):1011-1023.
155.Ishii K J,Weiss W R,Ichino M,et al.Activity and safety of DNAplasmids encoding Ik-4and IFN-gamma.Gene Ther,1999,6(2):237-244.
156.Vagner S,Galy B,Pyronnet S.Irresistible IRES.Attracting the translation machinery to internal ribosome entry sites.EMBO Rep,2001,2(10):893-898.
157.Molla A,Paul AV,Schmid M,et al.Studies on dicistronic polioviruses implicate viral proteinase 2A pro in RNA replication.Virololgy,1993,196(2):739.
158.Chen C Y,Sarnow P.Initiation of protein synthesis by eukaryotic translational apparatus on circular RNAs.Science,1995,268:415.
159.Niepmann M,Petersen A,Meyer K,et al.Functional involvement of polypyrimidine tract-binding protein in translation initiation complexes with the internal ribosome entry site of foot-and-mouth disease virus.J Virol,1997,71(11):8330-8339.
160.Kolupaeva V G,nellen C U,Shatsky I N.Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs.RNA,1996,2(12):1199-1212.
161.Mellen C U,Wimmer E.Translation of encephalomyocarditis virusRNA by internal ribosomal entry.Curr Top Microbiol Immunol,1995,203:31-63.
162.Gosert R,Chang K H,Rijnbrand R,et al.Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo.Mol Cell Biol,2000,20(5):1583-1595.
163.Iwasaki A,Stiernholm B J,Chan A K,et al.Enhanced CTL responses mediated by plasmid DNA immunogenes encoding costimulatory molecules and cytokines.J Immunol,1997,158(10):4591-4601.
164.Morris S,Kelley C,Howard A,et al.The immunogencity of single and combination DNA vaccines against tuberculosis,Vaccine,2000,18(20):2155-2163.
165.Turner O C,Roberts A D,Frank A A,et al.Lack of protection in mice and netrotizing bronchointerstitial pneumonia with bronchiolitis in guinea pigs immunized with vaccines directed against the hsp60 molecule of Mycobacterium tuberculosis.Infect Immunol,2000,68(6):3674-3679.
166.姜涛,于曼,范宝昌.等.我国登革2/4型病毒株PrM-E基因的双表达质粒构建及在真核细胞中的共表达.中国生物化学与分子生物学报,2003,19(3):278-282.
167.Boursnell M E C,Brow T D K,Foulds I J,et al.Completion of the sequence of the genome of the Coronavirus infectious bronchitis virus.J Gen Virol,1987,68:57-77.
168.Sterm D F.Kennedy S T.Coronavirus multiplication strategy I.Identification and characterization of virus intracellular RNA species to the genome.J Virol,1980:34(3):665-674.
169.殷震,刘景华主编.动物病毒学(第二版),北京:科学出版社,1997.
170.Brown A.J,Bracewell C D.Efect of repeated infectious of chickens with infectious bronchitis virus on the specificity of their antibodyer sponses.Vet Rec,1988,12(2):207-208.
171.Brown T D K,Boursnell M E G,Binns M M,et al.Cloning and sequencing of 5'terminal sequence from avian infectious bronchitis virus genomic RNA.J Gen Viral,1986,67:221-228.
172.江国托,刘思国,康丽娟,等.鸡传染性支气管炎病毒的遗传与变异(一).养禽与禽病防治,1999,(4):6-9
173.徐晓静,乌日罕,王立声,等.鸡传染性支气管炎分子生物学研究进展.畜牧与饲料科学,2005,26(2):28-30.
174.刘美丽,张兴晓,杨灵芝,等.禽传染性支气管炎病毒S基因的研究进展.动物医学进展,2004,25(5):15-17.
175.Gallagher T M.Murine coronavirus spike glycoprotein:Receptor binding and membrane fusion activities.Adv Exp Med Biol,2001,494:183-192.
176.Narayanan K,Maeda A,Maeda J,et al.Characterization of the Coronavirus M and Nucleocapsid Interaction in Infected Cells.J Virol,2000,74:8127-8134.
177.Cornelis A M H,Vennema H,Pottier P J.M.Assembly of the Coronavirus Envelope" Homotypic Interactions between the M protens.J.Virol,2000,.74:4967-4978.
178.Boots A M,Van Lierop M J,Kusters J G,et al.MHC class II-restrictedT-cell hybridomas recognizing the nucleocapsid protein of avian coronavirus IBV.Immunology,1991,72:10-14.
179.Boots A M,Kusters J G,van Noort J M,et al.Localization of a T-cell epitope within the nucleocapsid protein of avian coronavirus.Immunology,1991,74:8-13.
180.Emily Corse,Carolyn E,Machamer.Infectious bronchitis Virus E protein is targeted to the Golgi complex and directs release of virus-like particles.J Viorl,2000,74(9):4319-432.
181.Kim K H,Narayanan K,Makino S.Assembled coronavirus form complementation of two defective interfering RNAs.J Virol,1997,71:3922-3931.
182.De Haan,Shen X,weiss s,et al.The group-specific murine coronavirus genes are not essential,but their deletion,by reverse genetics,is attenuatin8 in the natural host.Virology,2002,296:177-189.
183.谢京国,方六荣,陈焕春等,双基因共表达的“自杀性”DNA疫苗载体的改造及其体外表达特性.中国兽医学报.2006,26(6):583-585.
184.Andreas Henke,Nadine Rohland,Roland Zell,et al.Co-expression of interleukin-2 by a bicistronic plasmid increases the efficacy of DNA immunization to prevent influenza virus infections.Intervirology,2006,49:249-252.
185.甘燕,吴少庭,秦莉等,SAILS冠状病毒E蛋白DNA疫苗的构建及其实验免疫研究.中国人兽共患病学报.2006,22(12):1139-1142.
2.苏敬良,高福译,Saif Y M主编.禽病学(第十一版),北京:中国农业出版社,2005.
3.Jia W,Karaca K,Parrish C R,et al.A novel variant of avian infectious bronchitis virus resulting from recombination among three different strains.Arch Virol,1995,140:259-271.
4.Wang H N,Wu Q Z,Huang Y,et al.Isolation and identification of infectious bronchitis virus from chickens in Sichuan,China.Avian Dis,1997,41:279-282.
5.Yu L,Jiang Y,Low S,et al.Characterization of three infectious bronchitis virus isolates from China associated with proventriculus in vaccinated chickens.Avian Dis,2001,45:416-424.
6.Liu S W,Kong X G.A new genotype of nephropathogenic infectious bronchitis virus circulating in vaccinated and nonvaccinated flocks in China.Avian Pathol,2004,33:321-327.
7.Zhou J Y,Zhang D Y,Ye J X,et ah Characterization of an avian infectious bronchitis virus isolated in China from chickens with nephritis.J Vet Med B Infect Dis Vet Public Health,2004,51:147-152.
8.Bayry J,Goudar M S,Nighot P K,et al.Emergence of a nephropathogenic avian infectious bronchitis virus with a novel genotype in India.J Clin Microbiol,2005,43:916-918.
9.Alvarado I R,Villegas P,Mossos N,et al.Molecular characterization of avian infectious bronchitis virus strains isolated in Colombia during 2003.Avian Dis,2005,49:494-499.
10.Ignjatovic J,Gould G,Sapats S.Isolation of a variant infectious bronchitis virus in Australia that further illustrates diversity among emerging strains.Arch Virol,2006,151:1567-1585.
11.江国托,刘思国,康丽娟,等.鸡传染性支气管炎病毒的遗传与变异(一).养禽与禽病防治,1999,(4):6-9.
12.Cavanagh D,Davis P J,Darybyshire J H,et al.Coronavirus IBV:virus retaining spike glycopolypeptide S2 but not S1 is unable to induce virus neutralizing or haemagglutination inhibiting antibody,or induce chicken tracheal protection.Journal of General Virology,1986,67(7):1435-1442.
13.Koch G,Kant A.Nucleotide and amino acid sequence of the S1 subunit of the spike glycoprotein of the coronavirus IBV.Nuc1 Acids Res,1990,18:3063-3064.
14.Collisson E W,Pei J,Dzielawa J,et al.Cytotoxic T lymphocytes are critical in the control of infectious bronchitis yirus in poultry.Dev Comp Immunol,2000,24:187-200.
15.Seo S H,Wang L,Smith R,et al.The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection.J Virol,1997,71,7889-94.
16.Mockett A P,Cavanagh D,Brown T D.Monoclonal antibodies to the S1 spike and membrane proteins of avian infectious bronchitis coronavirus strain Massachusetts M41.J Gen Virol,1984,65:2281-2286.
17.Mockett A P.Envelope proteins of avian infectious bronchitis virus:purification and biological properties.J Virol Methods,1985,12:271-278.
18.Klumperman J,Locker J K,Meijer A,et al.Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding.J Virol,1994,68:6523-6534.
19.Schalk A K,Hawn M C.An apparently new respiratory disease of baby chicks.J Am Vet Med Assoc,1931,78:413-422.
20.Capua I,Minta Z,Karpinska E,et al.Cocirculation of four types of infectious bronchitis virus(793/B 624/I B1648 and Massachusetts).Avian Pathol,1999,28:587-592.
21.CavanaghD,Ellis M M,Cook J K A.Relationship between sequence variation in the A1spike protein of infectious bronchitis virus and the extent of cross-protection in vivo.Avian Pathol,1997,26:63-74.
22.Cavanagh D,Severe acute respiratory syndrome vaccine development:experiences of vaccination against avian infectious bronchitis coronavirus.Avian Pathol,2003,32:567-582.
23.Cook J K A,Orbell S J,Woods M A,et al.Breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous types.Avian Pathol,1999,28:471-479.
24.Darbyshire J H,Rowell J G,Cook J K A,et al.Taxonomic studies on strains of avian infectious bronchitis virus using neutralisation tests in tracheal organ cultures.Arch Virol,1979,61:227-238.
25.De Wit J J.Detection of infectious bronchitis virus.Avian Pathol,2000,29:71-93.
26.El Houadfi M,Jones R C,Cook J K A,et al.The isolation and characterization of six avian infectious bronchitis viruses isolated in Morocco.Avian Pathol,1986,15:93-105.
27.Farsang A,Ros C,Renstrom L HM,et al.Molecular epizootiology of infectious bronchitis Virus in Sweden indicating the involvement of a vaccine strain.Avian Pathol,2002,31:229-236.
28.Gelb J Jr,Weisman Y,Ladman B S,et al.$1 gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the United States and Israel(1996 to 2000).Avian Pathol,2005,34:194-203.
29.Hofstad M S.Cross-immunity in chickens using seven isolates of avian infectious bronchitis virus.Avian Dis,1981,25:650-654.
30.Ladman B S,Loupos A B,Gelb J Jr.Infectious bronchitis virus S1 gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization.Avian Pathol,2006,35:127-133.
31.Lambrechts C,Pensaert M,Ducatelle R,Challenge experiments to evaluate crossprotection induced at the trachea and kidney level by vaccine strains and Belgian nephropathogenic isolates of avian infectious bronchitis virus.Avian Pathol,1993,22:577-590.
32.Picault J P,Drouin P,Guittet M,et al.Isolation characterisation and preliminary cross-protection studies with a new pathogenic avian infectious bronchitis virus(strain PL-84084).Avian Pathol,1986,15:367-383.
33.廖明,辛朝安,王林川,等.鸡传染性支气管病毒S1基因在昆虫细胞中的表达.中国兽医学报.1997,6(17):539-543.
34.宋延华,刘福安.鸡传染性支气管炎病毒S工基因在昆虫细胞中的高效表达.中国兽医学报,1999,19(5):421-424.
35.戴亚斌,陈德胜,丁铲,等.表达鸡传染性支气管炎病毒SD/97/01株S1蛋白的重组杆状病毒的构 建.厦门大学学报:自然科学版,2002,41(4):487-492.
36.Song C S,Lee Y J.Induction of protective immunity in chickens vaccinated with infectious bronchitis virus Slglycoprotein expressed by a recombinant baculovirus.J Gen Virol,1998,79(4):719-723.
37.步志高,江国托,王秀荣,等.传染性支气管炎病毒S1基因DNA免疫质粒的构建及其免疫原性.中国兽医学报,1998,18(6):527-530.
38.陈洪岩,江国托,杨奇伟,等.鸡传染性支气管炎病毒S1基因免疫对鸡的保护作用.中国预防兽医学报,1999,21(4):250-252.
39.江国托,步志高,刘思国,等.鸡传染性支气管炎病毒中国流行株免疫原S1基因的分子克隆、序列分析及其DNA免疫的初步研究.生物工程学报,1999,15(3):305-309.
40.Kapcynski D R.Protection of chickens from infectious bronchitis by in ovo and intramuscular vaccination with a DNA vaccine expressing the S1 glycoprotein.Avian Dis,2003,47:272-285.
41.刘思国,康丽娟.江国托,等.鸡传染性支气管炎病毒核蛋白基因重组真核表达载体的构建.中国兽医学报,2001,21(1):14-16.
42.李晓英,王红宁,黄勇,等.禽传染性支气管炎脂质体、壳聚糖/DNA疫苗免疫效果比较研究.畜牧兽医学报,2006,37(12):1319-1322.
43.Casais R,Thiel V,Siddell S G,et al.Reverse genetics system for the avian coronavirus infectious bronchitis virus.J Virol,2001,75:12359-12369.
44.Hodgson T,Casais R,Dove B,et al.Recombinant infectious bronchitis coronavirus Beaudette with the spike protein gene of the pathogenic M41 strain remains attenuated but induces protective immunity.J Virol,2004,78:13804-13811.
45.Tomley F M,Mockett A P,Boursnell M E,et al.Expression of theinfectious bronchitis virus spike protein by recombinant vaccinia virus and induction of neutralizing antibodies in vaccinated mice.J Gen Virol,1987,68:2291-2298.
46.Yu L,Liu W,Schnitzlein W M,et al.Study of protection by recombinant fowl poxvirus expressing C-terminal nucleocapsid protein of infectious bronchitis virus against challenge.Avian Dis,2001,45:340-348.
47.Wang X,Schnitzlein W M,Tripathy D N,et al.Construction and immunogenicity studies of recombinant fowl poxvirus containing the S1 gene of Massachusetts 41 strain of infectious bronchitis virus.Avian Dis,2002,46:831-838.
48.Johnson M A,Pooley C,Ignjatovic J,et al.A recombinant fowl adenovirus expressing the S1 gene of infectious bronchitis virus protects against challenge with infectious bronchitis virus.Vaccine,2003,21:2730-2736.
49.Zhou J Y,Cheng L Q,Zheng X J,et al.Generation of the transgenic potato expressing full-length spike protein of infectious bronchitis virus.J Biotechnol,2004,111:121-130.
50.李春,王红宁,周生,等.禽传染性支气管炎病毒S1基因玉米表达载体的构建.四川畜牧兽医,2003,30(B09):18-18.
51.Hildegund C J E.DNA疫苗.李琦涵,刘龙丁,车艳春,译.北京:化学工业出版社,2005.
52.房文亮,李泽民,揣侠,等.白细胞介素2和15基因对柯萨奇病毒VP1 DNA疫苗诱导免疫应答的影响.临床心血管病杂志,2005,21(1):33-35.
53.Min W,Lillehoj H S,et al.Adjuvant effects of IL-I beta,IL-2,IL-8,IL-15,IFN-alpha,IFN-gamma,TGF-beta4 and lymphotactin on DNA vaccinatiom against Eimeria acervulina.Vaccine,2001,20(1-2):267-274.
54.江文正.金宁一,李子健,等.IL-18和HIV-1 gag-sp120嵌合基因DNA疫苗联合免疫小鼠的免疫应答.细胞与分子免疫学杂志,2004,20(4):433-435.
55.Billaut-Mulot O,Idziorek T,Loyens M,et al.Modulation of cellular and humoral immune responses to a multiepitopic HIV-1 DNA vaccine by interleukin-18 DNA immunization/viral protein boost.Vaccine,2001,19(20-22):2803-2811.
56.Lid X J,Zhu M Z,Song G X,et al.Enhancement of herpes simplex virus-1 glycoprotein-D DNA vaccine induced specific immune responses by coimmunization with interleukin-2genetic adjuvant.Zhongguo Yi Xue Ke Xue Yuan Xue Bao,2005,27:67-72.
57.Barouch D H,Santra S,Steenbeke T D,et al.Augmentation and suppression of immune response to an HIV-1 DNA vaccine by plasmid cytokine/Ig administration.Immunol,1998,161:1875-1882.
58.程相朝,赵德明.鸡IL-18真核表达载体的构建及其对新城疫疫苗免疫增强作用的研究.畜牧兽医学报,2005,36(5):476-481.
59.Degen W G,van Zuilekom H I,Scholtes N C,et al.Potentiation of humoral immune responses to vaccine antigens by recombinant chicken IL-18(rChIL-18).Vaccine.2005,23(33):4212-4218.
60.Xiang Z Q,Spitalink K,Tran M,et al.Vaccinatiion with a plamid vetor carrying the rabies glycoprotein gene induces protective immunity against rabies virus.Virology,1994:132-140.
61.Barouch D H,Santra S,Racz P et al.Potent CD4+ T cell response elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF.Immunology,2002,168:562-568.
62.Wang J,GujarSA,Cova L,et al.Bicistronic woodchuck hepatitis virus core and gamma interferon DNA vaccine can protect from hepatitis but does not elicit sterilizing antiviral immunity.J Virol,2007,81:903-916.
63.Singh G,Parker S,Hobart P.The development of a bicistronic plasmid DNA vaccine for B-cell lymphoma.Vaccine,2002,31(20):1400-1411.
64.Mountford P S,Smith A G.Internal ribosome entry site and dicistronic RNAs in mammalian transgenesis.Trends Genet,1995,11(5):179-184.
65.Pelletier J,Sonenberg N.Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA.Nature,1988,334(6180):320-325.
66.Jang S K,Krausslich H G,Nicklin M J,et al.A segment of the 59 nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation.J Virol,1988,62(8):2636-2643.
67.詹林盛,饶林,彭剑淳,等.HCV IRES介导萤火虫荧光素酶翻译的双顺反子载体的构建与在小鼠体内的表达.中国生物化学与分子生物学报,2004.20(3):330-334.
68.Wild J,Gruner B,Metzger K,et al.Polyvalent vaccination against hepatitis B surface and core antigen using a dicistronic expression plasmid.Vaccine,1998,16:353-360.
69.郝敏麒,徐军,钟南山.尘瞒主要变应原DNA疫苗对其提取液诱导小鼠肺部变应性炎症的免疫保护作用.中华结核和呼吸杂志,2002,25:159-161.
70.Maecker H T,Hansen G,Walter D M,et al.Vaccination with allergen-IL-18 fusion DNA protects against and reverses established airway hyperreal livity in a murine asthma model.J Immunol.2001.166:959-965.
71.陈创大,余兴龙,马正海,等.细胞因子与猪瘟病毒E2基因真核表达载体的构建及其免疫增强作用.中国农业科学.2002,35(11):1408-1410.
72.Boursnell M E C,Brow T D K,Foulds I J,et al.Completion of the sequence of the genome of the Coronavirus infectious bronchitis virus.J Gen Virol,1987,68:57-77.
73.Liu D X,Cavanagh D,et al.A polycistronic mRNA specified by the coronavirus IBV.Virology,1991,184(2):531-544.
74.Jia W,Naqi S A.Sequence analysis of gene 3,gene 4 and gene 5 of avian infectious bronchitis virus strain CU-T2.Gene,1997,189(2):189-193.
75.Shen S,Wen Z L,Liu D X.Emergence of a cornavirus infectious bronchitis virus mutant with a truncated 3b gene:functional characterization of the 3b protein in pathogenesis and replication.Virology,2003,311:16-27.
76.Teri Hodgson,Poul Britton,Dave Cavanagh,et al.Neither the RNA nor the protein of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication.Virology,2006,80:296-305.
77.Liu D X,Inglis S C,etal.Internal entry of ribosomes on a tricistronic mRNA encoded by infectious bronchitis virus.Journal of Virology,1991,66(10):6143-6154.
78.Pelletier J,Sonenberg N.Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA.Nature,1988:320-334.
79.Jang S K,Krausslich H G,Nicklin M J,et al.A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosome during in vitro translation.J Virol,1988,62:2636.
80.Wang H N,Wu Q Z,nuang Y,et al.Isolation and identification of infectious bronchitis virus from chickens in Sichuan,China.Avian Dis,1997,41:279-282.
81.黄勇,王红宁,王林川,等.应用RT-PCR技术扩增禽传染性支气管炎病毒S1基因的研究.中国畜禽传染病,1998,20(5):297-298.
82.王林川,王红宁,黄勇,等.用RT-PCR和RFLP对禽传染性支气管炎病毒中国分离株的分型.病毒学报,1997,13(4):365-368.
83.余祖华,王红宁,周生,等.禽传染性支气管炎病毒S1基因毕赤酵母表达载体的构建.中国家禽,2006,28:14-16.
84.周生,王红宁,唐梦君,等.禽传染性支气管炎冠状病毒SAIBK株全基因组cDNA文库的构建与序列测定.畜牧兽医学报,2007,1:72-77.
85.Tahara H,Zitvogel L,Storkus W J,et al.Effective eradication of established murine tumors with IL-12 gene therapy using a polycistronic retroviral vector.J Immunol,1995,154(12):6466-6674.
86.David L,Suarez,Stacey Schultz-Cherry.The effect of eukaryotic expression vectors and adjuvants on DNA vaccines in chickens using an avian influenza model.Avian Disease,2000,44:861-868.
87.Alexander L,Lu H H,Gromeier M,Wimmer E.Dicistronic polioviruses as expression vectors for foreign genes.AIDS Res Hum Retorviruses,1994,10(2):57-60.
88.Pugachev K V,Tzeng W P,Frey T K.Development of a rubella virus vaccine expression vector:use of a picornavirus internal ribosome entry site increases stability of expression.J Virol,2000 Nov,74(22):10811-10815.
89.Benvenisti L,Rogel A,Kuznetzova L,et al.Gene gun-mediate DNA vaccination against foot-and-mouth disease virus.Vaccine,2001,19(28-29):3885-3895.
90.Osorio J E,Powell T D,Frank R S,etal.Recombinant raccoon pox vaccine portects mice against lethal plague.Vaccine,2003:21(11-12):1232-1238.
91.Lewis J C.Applications of reporter genes.Analytical Chemistry,1998,70(17):579-585.
92.薛丽香.童坦君,张宗玉,等.报告基因的选择及其研究趋向.生理科学进展,2002,33(4):364-366.
93.刘兆球,姜世金,常维山,等.传染性支气管炎病毒S1基因的真核表达及免疫原性检测.中国兽医学报,2005,25(3):241-243.
94.焦红梅,焦新安,殷月兰,等.鸡传染性支气管炎病毒S1基因的真核表达及其产物的免疫原性.扬州大学学报(农业与生命科学版),2006,27(2):44-47.
95.Ignjatovic J,Galli L.Immune responses to structural proteins of avian infectious virus.Avian Pathol,1995,24:313-332.
96.Borman A M,Mercier P L,Girard M,et al.Comparison of picornaviral IRES-driven internal initiation of translation in cultured cells of different origins.Nucleic Acids Res,1997,25(5):925-932.
97.Hiroyuki Mizuguchi,Zhili Xu,Akiko Ishii-Watabe,et al.IRES-Dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector.Molecular Ther,2000,1(4):376-382.
98.Saporito-Irwin S M,Geist R T,Gutmann D H.Ammonium acetate protocol for the preparation of plasmid DNA suitable for mammalian cell transfections.Biotechniques.1997,23(3):424-427.
99.姜世金,刘兆球,常维山,等.传染性支气管炎病毒S1基因真核表达质粒的构建及其在CEF中的转染.中国兽医杂志,2004,40(12):17-18.
100.赵宝华,马同锁,杨虹,等.鸡传染性支气管M41毒株S1基因的克隆与真核表达研究.动物医学进展,2005,26(1):71-75.
101.周生,王红宁.禽传染性支气管炎病毒分离株的全基因组及S1、M、N基因DNA疫苗研究.四川农业大学博士学位论文,2007.
102.陆彬.药物新剂型与新技术.北京:人民卫生出版社,1998,107-110.
103.Ishii N,Fukushima J,Kaneko T,et al.Cationic liposomes area strong adjuvant for a DNA vaccine of human immunodeficiency virus type.Aids Research in human retroviruses,1997,13:1421-1428.
104.彭保卫,郭祀远,杨富强,等.HBV DNA疫苗阳离子脂质体复合物转染小鼠实验研究.广东药学学院学报,2002.18(4):294-297.
105.Iqbalet M,Lin W,Jabbal Gill I,et al.Nasal delivery of chitosan-DNA plasmid expressing epitopes of respiratory syncytial virus(RSV)induces protective CTL responses in BALB/c mice.Vaccine,2003,21(13-14):1478.
106.Roy K,Mao H Q,Huang S K,et al.Oral gene delivery with chitosan-DNA nanoparticles generates immunologic protection in a murine peanut a11ergy model.Natmed,1999,5:387..
107.Cavanagh D.,Severe acute respiratory syndrome vaccine development:experiences of vaccination against avian infectious bronchitis coronavirus,Avian Pathol,2003,32:567-582.
108.De Wit J J.Detection of infectious bronchitis virus,Avian Pathol,2000,29:71-93.
109.Henke A,Rohland N,Zell R,et al.Co-expression of interleukin-2 by a bicistronic plasmid increases the efficacy of DNA immunization to prevent influenza virus infections.Intervirology,2006,49:249-52.
110.Henke A,Chiang C S,Zell R,et al.Co-expression of interleukin-2 to increase the efficacy of DNA vaccine-mediated protection in coxsackievirus B3-infected mice. Antiviral Res,2004,64:131-136.
111.Barouch D H,Santra S,Steenbeke T D et al.Augmentation and suppression of immune response to an HIV-1 DNA vaccine by plasmid cytokine/Ig administration.Immunol,1998,161:1875-1882.
112.Marshall D J,Rudnick K A,McCarthy S G,et al.Interleukin-18 enhances Th1 immunity and tumor protection of a DNA vaccine.Vaccine,2006,24:244-53.
113.Kim J J,Yang JS,Dang K,et al.Engineering enhanacement of immune responses to DNA-based vaccines in a prostate cancer model in rhesus macaques through the use of cytokine gene adjuvants.Clin Cancer Res,2001,7(Suppl 3):882-889.
114.Timms L M,Bracewell C D.Cell mediated and humoral immune response of chickens to live infectious bronchitis vaccines.Res Vet Sci,1981,31:182-189.
115.Chow Y H,Huang W L,Cli W K,et al.Improvement of hepatitis B virus DNA vaccine by plasmids coexpressing hepatitis B surface antigen and interleukin-2.J Virol,1997,71(1):169-178.
116.Li W R,Niu B,Wang J W,et al.Coexpression of interleukin-2 enhances the immunization effect of aDNAvaccine expressing herpes simplex 1 glycoprotein D:Acta Virol,2006,50:251-256.
117.Kamath A T,Hanke T,Briscoe H,et al.Co-immunization with DNA vaccines expressing granulocyte-macrophage colony-stimulating factor and mycobacterial secreted proteins enhances T-cell immunity,but not protective efficacy against Mycobacterium tuberculosis.Immunology,1999,96:511.
118.洪晓武,窦骏,陈国兵,等.mGM-CSF协同抗肿瘤作用研究进展.实用肿瘤杂志,2003,18:154.
119.Lgnjatovic J,Galli L.The S1 glycoprotein but not the N or M proteins of avian infectious bronchitis virus induces protection in vaccinated chickens.Arch Virol,1994,138:117-134.
120.Wang C H,Huang Y C.Relationship between serotypes and genotypes based on the hypervariable region of the S1 gene of infectious bronchitis virus.Arch Virol,2000,145:291-300.
121.Corse E,Machaser C E,The cytoplasmic tail of infectious bronchitis virus E protein directs Golgi targeting.J Virol,2002,76(3):1273-1284.
122.张德勇,周继勇.基因IBV重组表达蛋白的抗体检测技术及基因免疫研究.浙江大学博士学位论文,2005.
123.David F Stern,Bartholomer M Sefton.Coronavirus proteins:structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins.Virology,1982,804-812.
124.刘胜旺,贺秀瑗,孔宪刚,等.鸡传染性支气管炎病毒中国分离株M基因的分子特征.中国兽医学报,2001,23(6):404-407.
125.Ng Lisa F P,Liu D X,Identification of a 24-kDa polypeptide processed from the Coronavires infectious bronchitis virus 1a polyprotein by the 3c-like proteinase and determination of its cleavage sites.Virology,1998,243:388-395.
126.Lim Z P,Liu D X,The missing link in coronavirus assembly.Retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-Golgi compartments and physical interaction between the envelope and membrane proteins.J Biol Chem,2001,276(20):17515-17523.
127.Junko M,John F R,Akihiko M,et al.Rapid communication:Membrane topology of coronavirus E protein.Virology,2001,281(i):163-169.
128.Dave cavanagh.Coronavirus avian infectious bronchitis virus.Vet Res,2007,38:281-297.
129.黄亚东,王林川,郑青,等.鸡传染性支气管炎病毒H株纤突蛋白S1基因的克隆及其在毕赤酵母中表达.病毒学报,2003,19(2):144-148.
130.周继勇,JimmyKwang.传染性支气管炎病毒核衣壳蛋白基因变异及在大肠杆菌中的表达.浙江大学学报:农业与生命科学版,2003,29(1):1-9.
131.刘胜旺,杜恩歧,孔宪刚,等.鸡传染性支气管炎病毒核蛋白基因的体外表达和纯化.中国实验动物学杂志,2002,12(5):302-302.
132.陈萍,王红宁.间接ELISA检测IBV DNA疫苗免疫抗体及IBV M基因表达研究,四川农业大学硕士论文,2005
133.Binns M M,et al.Israel Journal of veterinary Medicine,1986,42(2):124-127.
134.Evans S,Cavanagh D.Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus.J Gem Virol,2000,81(12):2855-2865.
135.Yu L,Liu W,Schnitzlein W M,et al.Study of protection by recombinant fowl poxvirus expressing C-terminal nucleocapsid protein of infectious bronchitis virus against challenge.Avian Dis,2001,45:340-348.
136.田占成,孙永科,王云峰,等.表达鸡传染性支气管炎病毒S1基因重组鸡痘病毒对SPF鸡的免疫保护作用.畜牧兽医学报,2006,37(6):580-586.
137.焦红梅,焦新安,田华荣,等.减毒鼠伤寒沙门氏菌运送的鸡传染性支气管炎病毒S1基因DNA疫苗及其免疫效力研究.病毒学报,2007,23(3):212-117.
138.Kapcynski D R.Protection of chickens from infectious bronchitis by in ovo and intramuscular vaccination with a DNA vaccine expressing the S1 glycoprotein.Avian Dis,2003,47:272-285.
139.杨帆,王红宁,李春,等.禽传染性支气管炎病毒四川分离株M基因的克隆及真核表达载体构建。四川畜牧兽医,2003,30(B09):19-19.
140.Xin K Q,Hamajima K,Sasar S,et al.Intranasal administration of human immunodeficiency virus type 1(HIV1)DNA vaccine with interleukin2 expression plasmid enhance cell mediated immunity against HIV.Immunology,1998,94(3):438-444.
141.Kim J J,Ayyavoo V,Bagarass M L,et al.In vivo engineering of a cellular immune response by coadministration of IL-12 expression vetor with a DNA immunogen.J Immunol,1997,158:816-826.
142.Gherardi M M,Ramirez J C.Esteban M.Towards a new generation of vaccine:the cytokine IL-12 as an adjuvant to enhance cellular inmmune responses to pathogens during prime-booster vaccination regimens.Histol Histopathol,2001,16(2):655-667
143.Okada H,Giezeman-Smits K M,Tahara H,et al.Effective cytokine gene therapy against an intracranial glioma using a retrovirally transduced IL-4 plus HSV tk tumor vaccine.Gene Ther,1999,6(2):219-226.
144.Kim S H,Cho D,Hwang S Y,et al.Efficient induction of antigenspecific T helper type Ⅰ mediated immune responses by intramuscular injection with ovalbumin/interleukin-18fusion DNA.Vaccine,2001,19(30):4107-4114.
145.Sin J I,Kim J J,Boyer J D,et al.In vivo modulation of vaccine induced immune responses toword a Th1 phenotype increases potency and vaccine effectiveness in herpes simplex virus type 2 mouse model.J Virol,1999,73(1):501-509.
146.Geissler M,Gesien A,Tokushige K,et al.Enhancement of cellular and hunoral immune responses to hepatitis C titus core protein using DNA-based vaccinges augmented with cytokine-expressing plasmids.J Immunol,1997,158:1231-1237.
147.Kim J J,Yang J S,Manson K M,et al.Coimmunization with IFN-gamma or IL-2,but Not IL-13 or IL-4 cDNA Can Enhance Th1-Type DNA Vaccine-Induced.Journal of Interferon &Cytokine Research,2000,20(3):311-319.
148.Min W,Lillehoj H S,et al.Adjuvant effects of IL-1 beta,IL-2,IL-8,IL-15,IFN-alpha,IFN-gamma,TGF-beta4 and lymphotactin on DNA vaccinatiom against Eimeria acervulina.Vaccine,2001,20(1-2):267-274.
149.Lim Y S,Kang B Y,Kim E J,et al.Potentiation of antigen-specific,T hi immune response by multiple DNA vaccination with an ovalbumin/interferon-γ hybrid construct.Immunol,1998,94:135-141.
150.Mbawuike I N.Enhancement of the protecting efficacy of inactivated influenza A vaccine in aged mice by IL-2-1iposomes.Vaccine,1990,8:347-352.
151.Operschall E,SchuhT,Heinzerling L,et al.Enhanced protection against viral infection by co-administration of plasmid DNA coding for viral antigen and cytokines in mice.J Clin Virol,1999,13:17-22.
152.杜德伟,周永兴,冯志华,等.IL-12及HBV基因疫苗共同免疫小鼠的效果.世界华人消化杂志,2000,8(2):128-130.
153.Lee S W,Cho J M,Sung Y C,et al.Optimal induction of hepatitis C virusenvelope-specific immunity by bicistronic plasmid DNA inoculation with thegranulocyte-macrophage colony-stimulating factor gene.J Virol,1998,72:8430-8436.
154.Parker S E,Monteith D,.Morton H,et al.Safety of a GM-CSF adjuvant-plasid DNAmalaria vaccine.Gene Ther,2001,8(13):1011-1023.
155.Ishii K J,Weiss W R,Ichino M,et al.Activity and safety of DNAplasmids encoding Ik-4and IFN-gamma.Gene Ther,1999,6(2):237-244.
156.Vagner S,Galy B,Pyronnet S.Irresistible IRES.Attracting the translation machinery to internal ribosome entry sites.EMBO Rep,2001,2(10):893-898.
157.Molla A,Paul AV,Schmid M,et al.Studies on dicistronic polioviruses implicate viral proteinase 2A pro in RNA replication.Virololgy,1993,196(2):739.
158.Chen C Y,Sarnow P.Initiation of protein synthesis by eukaryotic translational apparatus on circular RNAs.Science,1995,268:415.
159.Niepmann M,Petersen A,Meyer K,et al.Functional involvement of polypyrimidine tract-binding protein in translation initiation complexes with the internal ribosome entry site of foot-and-mouth disease virus.J Virol,1997,71(11):8330-8339.
160.Kolupaeva V G,nellen C U,Shatsky I N.Structural analysis of the interaction of the pyrimidine tract-binding protein with the internal ribosomal entry site of encephalomyocarditis virus and foot-and-mouth disease virus RNAs.RNA,1996,2(12):1199-1212.
161.Mellen C U,Wimmer E.Translation of encephalomyocarditis virusRNA by internal ribosomal entry.Curr Top Microbiol Immunol,1995,203:31-63.
162.Gosert R,Chang K H,Rijnbrand R,et al.Transient expression of cellular polypyrimidine-tract binding protein stimulates cap-independent translation directed by both picornaviral and flaviviral internal ribosome entry sites In vivo.Mol Cell Biol,2000,20(5):1583-1595.
163.Iwasaki A,Stiernholm B J,Chan A K,et al.Enhanced CTL responses mediated by plasmid DNA immunogenes encoding costimulatory molecules and cytokines.J Immunol,1997,158(10):4591-4601.
164.Morris S,Kelley C,Howard A,et al.The immunogencity of single and combination DNA vaccines against tuberculosis,Vaccine,2000,18(20):2155-2163.
165.Turner O C,Roberts A D,Frank A A,et al.Lack of protection in mice and netrotizing bronchointerstitial pneumonia with bronchiolitis in guinea pigs immunized with vaccines directed against the hsp60 molecule of Mycobacterium tuberculosis.Infect Immunol,2000,68(6):3674-3679.
166.姜涛,于曼,范宝昌.等.我国登革2/4型病毒株PrM-E基因的双表达质粒构建及在真核细胞中的共表达.中国生物化学与分子生物学报,2003,19(3):278-282.
167.Boursnell M E C,Brow T D K,Foulds I J,et al.Completion of the sequence of the genome of the Coronavirus infectious bronchitis virus.J Gen Virol,1987,68:57-77.
168.Sterm D F.Kennedy S T.Coronavirus multiplication strategy I.Identification and characterization of virus intracellular RNA species to the genome.J Virol,1980:34(3):665-674.
169.殷震,刘景华主编.动物病毒学(第二版),北京:科学出版社,1997.
170.Brown A.J,Bracewell C D.Efect of repeated infectious of chickens with infectious bronchitis virus on the specificity of their antibodyer sponses.Vet Rec,1988,12(2):207-208.
171.Brown T D K,Boursnell M E G,Binns M M,et al.Cloning and sequencing of 5'terminal sequence from avian infectious bronchitis virus genomic RNA.J Gen Viral,1986,67:221-228.
172.江国托,刘思国,康丽娟,等.鸡传染性支气管炎病毒的遗传与变异(一).养禽与禽病防治,1999,(4):6-9
173.徐晓静,乌日罕,王立声,等.鸡传染性支气管炎分子生物学研究进展.畜牧与饲料科学,2005,26(2):28-30.
174.刘美丽,张兴晓,杨灵芝,等.禽传染性支气管炎病毒S基因的研究进展.动物医学进展,2004,25(5):15-17.
175.Gallagher T M.Murine coronavirus spike glycoprotein:Receptor binding and membrane fusion activities.Adv Exp Med Biol,2001,494:183-192.
176.Narayanan K,Maeda A,Maeda J,et al.Characterization of the Coronavirus M and Nucleocapsid Interaction in Infected Cells.J Virol,2000,74:8127-8134.
177.Cornelis A M H,Vennema H,Pottier P J.M.Assembly of the Coronavirus Envelope" Homotypic Interactions between the M protens.J.Virol,2000,.74:4967-4978.
178.Boots A M,Van Lierop M J,Kusters J G,et al.MHC class II-restrictedT-cell hybridomas recognizing the nucleocapsid protein of avian coronavirus IBV.Immunology,1991,72:10-14.
179.Boots A M,Kusters J G,van Noort J M,et al.Localization of a T-cell epitope within the nucleocapsid protein of avian coronavirus.Immunology,1991,74:8-13.
180.Emily Corse,Carolyn E,Machamer.Infectious bronchitis Virus E protein is targeted to the Golgi complex and directs release of virus-like particles.J Viorl,2000,74(9):4319-432.
181.Kim K H,Narayanan K,Makino S.Assembled coronavirus form complementation of two defective interfering RNAs.J Virol,1997,71:3922-3931.
182.De Haan,Shen X,weiss s,et al.The group-specific murine coronavirus genes are not essential,but their deletion,by reverse genetics,is attenuatin8 in the natural host.Virology,2002,296:177-189.
183.谢京国,方六荣,陈焕春等,双基因共表达的“自杀性”DNA疫苗载体的改造及其体外表达特性.中国兽医学报.2006,26(6):583-585.
184.Andreas Henke,Nadine Rohland,Roland Zell,et al.Co-expression of interleukin-2 by a bicistronic plasmid increases the efficacy of DNA immunization to prevent influenza virus infections.Intervirology,2006,49:249-252.
185.甘燕,吴少庭,秦莉等,SAILS冠状病毒E蛋白DNA疫苗的构建及其实验免疫研究.中国人兽共患病学报.2006,22(12):1139-1142.