新型生长抑素DNA疫苗免疫猪的效果及其影响因素和作用机制研究
|
摘要
本研究应用基因免疫、酶联免疫测定、活体成像和石蜡切片等技术,将新型生长抑素DNA疫苗——减毒鼠伤寒沙门氏菌株CS022(pGM-CSF/SS)口服和鼻腔免疫仔猪,探讨其免疫效力及影响因素；另将减毒猪霍乱沙门氏菌株C500(pGS/2SS-M4GFP-asd)肌肉注射小鼠,探讨外源目的基因在组织器官中表达与分布的时空变化规律,为优化新型DNA疫苗的免疫程序,提高其对仔猪的促生长效果,加快其临床应用奠定基础。1.仔猪口服新型生长抑素DNA疫苗的免疫应答及其促生长效果
为了探索疫苗能否诱导仔猪产生免疫应答反应及其促生长效果,将40只9周龄的杜-长-大三元杂交猪随机分为4组,每组10只,公母各半。第1-3组(T1-T3)经口灌服低(5×108CFU/只)、中(5×109CFU/只)、高(5×1010CFU/只)三个剂量的减毒鼠伤寒沙门氏菌株CS022(pGM-CSF/SS)生长抑素DNA疫苗,第4组灌服5ml PBS溶液,用做对照(C1)。1W后以相同方式和剂量加强免疫1次。分别在免疫前、初免后4W、初免后8W称重3次；于免疫前及免疫后5W2次前腔静脉采集血样,用间接ELISA法检测血浆IgG抗体水平,用放射免疫法检测血浆中的SS浓度。
结果显示,免疫后仔猪行为正常,受试猪血浆中均能检测到SS抗体,以T3组抗体水平最高；与C1组相比,T1组(P>0.05)差异不显著,T2组(p<0.01)差异极显著,T3组(p<0.01)差异极显著。T3组与T1组相比(p<0.01),差异极显著。试验各组出现的免疫阳性猪比例,其中T1、T2组均为10.0%,T3组为40.0%。试验各组血浆SS浓度与免疫前和C1组相比均大大降低(P<0.01),差异极显著,但组间差异不显著。
免疫后0-4W,仔猪日增重T3组比C1组提高20.69%(P<0.05),差异显著,比T1组提高27.59%(P<0.01),差异极显著,比T2提高13.79%(P>0.05),差异不显著；T2组分别比C1组、T1组提高6.90%、13.80%(P>0.05),差异不显著。免疫后5-8W,仔猪日增重T1组比C1组提高11.11%(P>0.05),T2组和T3组比C1组均提高2.47%(P>0.05),差异不显著。免疫后0～8W,仔猪平均日增T3组比C1、T2、T1组分别提高10.00%、5.71%、7.14%(P>0.05),差异不显著。
以上结果表明,新型生长抑素DNA疫苗可诱导仔猪产生高水平免疫应答反应,有效促进了仔猪生长；免疫后仔猪SS抗体水平、抗体阳性猪所占比率和平均日增重均与免疫剂量存在正相关关系。
2.仔猪鼻腔免疫新型生长抑素DNA疫苗的促生长效果
为了探索鼻腔免疫新型生长抑素DNA疫苗对仔猪的促生长效果,将75只遗传背景、年龄一致,体重21.0±2.0Kg杜长大三元杂交阉公猪分成4组,前3组(T4-T6)每组20只,鼻腔免疫3次,每次间隔4W,剂量分别为低(2×108CFU/只)、中(2×109CFU/只)、高(2×1010CFU/只),第4组(C2)15只,鼻腔给药PBS溶液,剂量为2ml,用做对照。分别在免疫前、免疫后12W称量体重。结果显示,3个免疫组(T4-T6)平均日增重较对照组(C2)分别提高11.11%、6.17%、1.23%(P>0.05),差异不显著。随着免疫剂量的升高,平均日增重呈下降趋势,表明鼻腔免疫效果与剂量之间存在负相关关系。在日增重提升幅度一致情况下,鼻腔免疫需要的疫苗剂量远远少于口服免疫。
3.新型生长抑素DNA疫苗肌肉注射小鼠后目的基因表达与蛋白分布的时空变化规律
为了探索肌肉注射新型生长抑素DNA疫苗小鼠器官组织SS融合蛋白的时空变化规律,将78只7周龄昆明小鼠随机分为13组,每组6只,雌雄各半,采用肌肉纵向两点注射法在小鼠2个后肢股四头肌接种,剂量为150μL×109CFU/只。6个免疫组(A1～A6)免疫减毒猪霍乱沙门氏菌C500(pGS/2SS-M4GFP-asd)生长抑素DNA疫苗,6个阴性对照组(B1-B6)免疫不含质粒的C500空菌疫苗,1个组注射150μLPBS溶液,用于空白对照(C3)。分别于免疫后24h、48h、96h、144h、192h、240h6个时间点依次用乙醚熏晕处死A1～A6和B1-B6组的6只小鼠,C4组小鼠于注射24h处死,采集心脏、肝脏、脾脏、肺脏、肾脏和后腿肌肉样品制作石蜡切片,并用免疫组化法染色切片。
结果显示,仅在注射处肌肉、脾脏、肝脏组织中的巨噬细胞依次表达SS蛋白,并在144h到达峰值。肌肉组织最先(24h)表达,持续时间最长,240h仍有大量SS蛋白；脾脏次之,免疫48h开始表达,持续时间居中,240h只有少量SS蛋白；肝脏最晚(144h)表达,持续时间最短,240h已观察不到SS蛋白。结果表明肌肉注射免疫是可行的,外源DNA质粒在动物体内存留和目的蛋白持续表达的时间短暂,在家畜生产中可以应用肌肉注射方式免疫生长抑素DNA疫苗。
In this study, the immune efficacy and influence factors of a novel somatostatin DNA vaccine-Attenuated Mouse Typhus Salmonella strains CS022(pGM-CSF/SS) immunized against piglets with oral administration and nasal feeding were investigated, using methods of genetic immunization, ELISA, and paraffin section. Mice were administered attenuated Cholera Swine Salmonella strains C500(pGS/2SS-M4GFP-asd) through intramuscular injection to detect the temporal and spatial patterns of the exogenous gene expression in various tissues and organs, which will contribute to immunization program optimization of novel growth-promoting DNA vaccine, elevation of their growth-promoting effects on the piglets and to accelerate its clinical application.1. Immune responses and Growth-promoting effect of piglets to the novel
somatostatin DNA vaccine by oral vaccination. So as to study the immune responses and the growth-promoting effect of the novel somatostatin DNA vaccine by oral way. a total of40three-ways crossing commercial piglets at9weeks of age were randomly divided into4groups, and each group was evenly composed of5males and5females. T1, T2and T3groups were administered with attenuated Salmonella strain CS022(pGM-CSF/SS) somatostatin DNA vaccine orally with low doses (5×108CFU/each), moderate doses (5×109CFU/each), and high doses (5×1010CFU/each) respectively. Control group C1was administered with5ml PBS solution. A booster was performed with the same doses and same treatment at1week after primary immunization. Body weight gains of the piglets were measured three times (before immunization, after immunization4and8weeks). The blood samples were collected two times at former vena cava (before immunization and after immunization5weeks). IgG antibody levels in plasma was detected using indirect ELISA and SS concentration in plasma was detected using radioimmunoassay.
The results showed that the piglets had normal behavior after vaccination. Plasma SS antibodies were detected in all experimental groups, T3group had the highest level. Compared with C2group, T2group did not have significant difference (P>0.05) but both T2and T3had very significant difference (P<0.01). The immunoreactive pigs occurred in each experimental group, and the ratio of T1and T2group were10.0%and T3group was40.0%. Compared with those of pre-immunization and the Cl group, plasma SS concentration declined significantly for each group (p<0.01), but there was no significant difference among experimental groups (P>0.05).
T3group pigs average daily gain increased by20.69%compared with Cl group pigs (P<0.05), it had significant difference, and increasing by27.59%compared with T1group pigs (P<0.01) which it had significantly difference after immunization0-4weeks, but it increased only by13.79%compared with T2that had no significant difference. There was no significant difference (P>0.05) among all groups though T1group pigs average daily gain increased by11.11%compared with C1control group after immunization5-8weeks, and also T3group pigs average daily gain increased only by10.00%compared with C1control group after immunization0-8weeks.
These results demonstrated that the vaccine could induce the immune response in piglets and the immune effects were significant. There was positive correlation-dependent among antibody levels and the ratio of antibody postive piglets and average daily gain and dose amount.
2. Growth-promoting effect of novel somatostatin DNA vaccine by nasal injection
So as to study the growth-promoting effect of the novel somatostatin DNA vaccine by nasal injection, a total of75three-ways crossing commercial castrated male piglets with the same genetic background and weight21.0±2.0Kg, were divided into four groups. There were20pigs for each group of T4, T5and T6, and15for C2group. The pigs were administered vaccine3times at intervals of4weeks through nasal injection, with low dose (2×108CFU/each), moderate dose (2×109CFU/each) and high doses (2×1010CFU/each) respectively. C2group was treated with PBS solution with a dose of2ml. Body weight gains of the piglets were measured twice (before immunization and after immunization12weeks). The results showed that T4(low dose) group pigs average daily gain increased by11.11%compared with C2control group after immunization, T5(middle dose) and T6(high dose) group increased by6.17%and1.23%respectively. There was no significant difference (P>0.05) between groups. Average daily gain declined with the increasing dose of vaccine, which implied that immune efficacy had negative correlation with vaccine dose. Nasal immunization needed vaccine doses far less than oral immunization for the same daily gain.
3. The temporal and spatial patterns of the target gene and the SS fusion protein of immunized mice through intramuscular injection with novel somatostatin DNA vaccine
So as to study the temporal and spatial patterns of the target gene and the SS fusion protein of immunized mice through intramuscular injection with novel somatostatin DNA vaccine, a total of78Kunming mice at7weeks old were randomly divided into13groups, and each group was evenly composed of3males and3females. Longitudinal muscle injection with dose of150μL×109CFU/each at two hind quadriceps in mice was implemented. The immunized groups A1-A6were inoculated attenuated Salmonella choleraesuis C500(pGS/2SS-M4GFP-asd) somatostatin DNA vaccine, and negative control group B1-B6were inoculated with plasmid-free C500bacteria vaccine, and the control group C3inoculated with150μL PBS saline. Six mice of each group were killed after Ethyl ether smoked dizzy in turn with ether A1-A6and B1-B6at immunization24h,48h,96h,144h,192h and240h, and C3group mice was killed by the same way at immunization24h. Paraffin sections were made after collecting their heart, liver, spleen, lung, kidney and hindlimb skeletal muscle, and to deal with by Immunohistochemical staining method.
The results showed that SS fusion protein had been expressed in turn with at macrophage of spleen, liver, muscle with injection site, and its expression reaches the maximum all of them at immunization144h. It occurred firstly at muscle tissue (immunization24h), the second at spleen (immunization48h), the last at liver (immunization144h). Muscle tissue had sustained more time than spleen and liver, and had more quantity until immunization240h. Liver had the shortest time of continuous expression, and there was no SS fusion protein at immunization240h.
It demonstrated that it was viable to immune livestock the way of intramuscular injection with novel somatostatin DNA vaccine. It was short time that the exogenous plasmid DNA and purpose protein had continuous expression in mice by intramuscular injection.
为了探索疫苗能否诱导仔猪产生免疫应答反应及其促生长效果,将40只9周龄的杜-长-大三元杂交猪随机分为4组,每组10只,公母各半。第1-3组(T1-T3)经口灌服低(5×108CFU/只)、中(5×109CFU/只)、高(5×1010CFU/只)三个剂量的减毒鼠伤寒沙门氏菌株CS022(pGM-CSF/SS)生长抑素DNA疫苗,第4组灌服5ml PBS溶液,用做对照(C1)。1W后以相同方式和剂量加强免疫1次。分别在免疫前、初免后4W、初免后8W称重3次；于免疫前及免疫后5W2次前腔静脉采集血样,用间接ELISA法检测血浆IgG抗体水平,用放射免疫法检测血浆中的SS浓度。
结果显示,免疫后仔猪行为正常,受试猪血浆中均能检测到SS抗体,以T3组抗体水平最高；与C1组相比,T1组(P>0.05)差异不显著,T2组(p<0.01)差异极显著,T3组(p<0.01)差异极显著。T3组与T1组相比(p<0.01),差异极显著。试验各组出现的免疫阳性猪比例,其中T1、T2组均为10.0%,T3组为40.0%。试验各组血浆SS浓度与免疫前和C1组相比均大大降低(P<0.01),差异极显著,但组间差异不显著。
免疫后0-4W,仔猪日增重T3组比C1组提高20.69%(P<0.05),差异显著,比T1组提高27.59%(P<0.01),差异极显著,比T2提高13.79%(P>0.05),差异不显著；T2组分别比C1组、T1组提高6.90%、13.80%(P>0.05),差异不显著。免疫后5-8W,仔猪日增重T1组比C1组提高11.11%(P>0.05),T2组和T3组比C1组均提高2.47%(P>0.05),差异不显著。免疫后0～8W,仔猪平均日增T3组比C1、T2、T1组分别提高10.00%、5.71%、7.14%(P>0.05),差异不显著。
以上结果表明,新型生长抑素DNA疫苗可诱导仔猪产生高水平免疫应答反应,有效促进了仔猪生长；免疫后仔猪SS抗体水平、抗体阳性猪所占比率和平均日增重均与免疫剂量存在正相关关系。
2.仔猪鼻腔免疫新型生长抑素DNA疫苗的促生长效果
为了探索鼻腔免疫新型生长抑素DNA疫苗对仔猪的促生长效果,将75只遗传背景、年龄一致,体重21.0±2.0Kg杜长大三元杂交阉公猪分成4组,前3组(T4-T6)每组20只,鼻腔免疫3次,每次间隔4W,剂量分别为低(2×108CFU/只)、中(2×109CFU/只)、高(2×1010CFU/只),第4组(C2)15只,鼻腔给药PBS溶液,剂量为2ml,用做对照。分别在免疫前、免疫后12W称量体重。结果显示,3个免疫组(T4-T6)平均日增重较对照组(C2)分别提高11.11%、6.17%、1.23%(P>0.05),差异不显著。随着免疫剂量的升高,平均日增重呈下降趋势,表明鼻腔免疫效果与剂量之间存在负相关关系。在日增重提升幅度一致情况下,鼻腔免疫需要的疫苗剂量远远少于口服免疫。
3.新型生长抑素DNA疫苗肌肉注射小鼠后目的基因表达与蛋白分布的时空变化规律
为了探索肌肉注射新型生长抑素DNA疫苗小鼠器官组织SS融合蛋白的时空变化规律,将78只7周龄昆明小鼠随机分为13组,每组6只,雌雄各半,采用肌肉纵向两点注射法在小鼠2个后肢股四头肌接种,剂量为150μL×109CFU/只。6个免疫组(A1～A6)免疫减毒猪霍乱沙门氏菌C500(pGS/2SS-M4GFP-asd)生长抑素DNA疫苗,6个阴性对照组(B1-B6)免疫不含质粒的C500空菌疫苗,1个组注射150μLPBS溶液,用于空白对照(C3)。分别于免疫后24h、48h、96h、144h、192h、240h6个时间点依次用乙醚熏晕处死A1～A6和B1-B6组的6只小鼠,C4组小鼠于注射24h处死,采集心脏、肝脏、脾脏、肺脏、肾脏和后腿肌肉样品制作石蜡切片,并用免疫组化法染色切片。
结果显示,仅在注射处肌肉、脾脏、肝脏组织中的巨噬细胞依次表达SS蛋白,并在144h到达峰值。肌肉组织最先(24h)表达,持续时间最长,240h仍有大量SS蛋白；脾脏次之,免疫48h开始表达,持续时间居中,240h只有少量SS蛋白；肝脏最晚(144h)表达,持续时间最短,240h已观察不到SS蛋白。结果表明肌肉注射免疫是可行的,外源DNA质粒在动物体内存留和目的蛋白持续表达的时间短暂,在家畜生产中可以应用肌肉注射方式免疫生长抑素DNA疫苗。
In this study, the immune efficacy and influence factors of a novel somatostatin DNA vaccine-Attenuated Mouse Typhus Salmonella strains CS022(pGM-CSF/SS) immunized against piglets with oral administration and nasal feeding were investigated, using methods of genetic immunization, ELISA, and paraffin section. Mice were administered attenuated Cholera Swine Salmonella strains C500(pGS/2SS-M4GFP-asd) through intramuscular injection to detect the temporal and spatial patterns of the exogenous gene expression in various tissues and organs, which will contribute to immunization program optimization of novel growth-promoting DNA vaccine, elevation of their growth-promoting effects on the piglets and to accelerate its clinical application.1. Immune responses and Growth-promoting effect of piglets to the novel
somatostatin DNA vaccine by oral vaccination. So as to study the immune responses and the growth-promoting effect of the novel somatostatin DNA vaccine by oral way. a total of40three-ways crossing commercial piglets at9weeks of age were randomly divided into4groups, and each group was evenly composed of5males and5females. T1, T2and T3groups were administered with attenuated Salmonella strain CS022(pGM-CSF/SS) somatostatin DNA vaccine orally with low doses (5×108CFU/each), moderate doses (5×109CFU/each), and high doses (5×1010CFU/each) respectively. Control group C1was administered with5ml PBS solution. A booster was performed with the same doses and same treatment at1week after primary immunization. Body weight gains of the piglets were measured three times (before immunization, after immunization4and8weeks). The blood samples were collected two times at former vena cava (before immunization and after immunization5weeks). IgG antibody levels in plasma was detected using indirect ELISA and SS concentration in plasma was detected using radioimmunoassay.
The results showed that the piglets had normal behavior after vaccination. Plasma SS antibodies were detected in all experimental groups, T3group had the highest level. Compared with C2group, T2group did not have significant difference (P>0.05) but both T2and T3had very significant difference (P<0.01). The immunoreactive pigs occurred in each experimental group, and the ratio of T1and T2group were10.0%and T3group was40.0%. Compared with those of pre-immunization and the Cl group, plasma SS concentration declined significantly for each group (p<0.01), but there was no significant difference among experimental groups (P>0.05).
T3group pigs average daily gain increased by20.69%compared with Cl group pigs (P<0.05), it had significant difference, and increasing by27.59%compared with T1group pigs (P<0.01) which it had significantly difference after immunization0-4weeks, but it increased only by13.79%compared with T2that had no significant difference. There was no significant difference (P>0.05) among all groups though T1group pigs average daily gain increased by11.11%compared with C1control group after immunization5-8weeks, and also T3group pigs average daily gain increased only by10.00%compared with C1control group after immunization0-8weeks.
These results demonstrated that the vaccine could induce the immune response in piglets and the immune effects were significant. There was positive correlation-dependent among antibody levels and the ratio of antibody postive piglets and average daily gain and dose amount.
2. Growth-promoting effect of novel somatostatin DNA vaccine by nasal injection
So as to study the growth-promoting effect of the novel somatostatin DNA vaccine by nasal injection, a total of75three-ways crossing commercial castrated male piglets with the same genetic background and weight21.0±2.0Kg, were divided into four groups. There were20pigs for each group of T4, T5and T6, and15for C2group. The pigs were administered vaccine3times at intervals of4weeks through nasal injection, with low dose (2×108CFU/each), moderate dose (2×109CFU/each) and high doses (2×1010CFU/each) respectively. C2group was treated with PBS solution with a dose of2ml. Body weight gains of the piglets were measured twice (before immunization and after immunization12weeks). The results showed that T4(low dose) group pigs average daily gain increased by11.11%compared with C2control group after immunization, T5(middle dose) and T6(high dose) group increased by6.17%and1.23%respectively. There was no significant difference (P>0.05) between groups. Average daily gain declined with the increasing dose of vaccine, which implied that immune efficacy had negative correlation with vaccine dose. Nasal immunization needed vaccine doses far less than oral immunization for the same daily gain.
3. The temporal and spatial patterns of the target gene and the SS fusion protein of immunized mice through intramuscular injection with novel somatostatin DNA vaccine
So as to study the temporal and spatial patterns of the target gene and the SS fusion protein of immunized mice through intramuscular injection with novel somatostatin DNA vaccine, a total of78Kunming mice at7weeks old were randomly divided into13groups, and each group was evenly composed of3males and3females. Longitudinal muscle injection with dose of150μL×109CFU/each at two hind quadriceps in mice was implemented. The immunized groups A1-A6were inoculated attenuated Salmonella choleraesuis C500(pGS/2SS-M4GFP-asd) somatostatin DNA vaccine, and negative control group B1-B6were inoculated with plasmid-free C500bacteria vaccine, and the control group C3inoculated with150μL PBS saline. Six mice of each group were killed after Ethyl ether smoked dizzy in turn with ether A1-A6and B1-B6at immunization24h,48h,96h,144h,192h and240h, and C3group mice was killed by the same way at immunization24h. Paraffin sections were made after collecting their heart, liver, spleen, lung, kidney and hindlimb skeletal muscle, and to deal with by Immunohistochemical staining method.
The results showed that SS fusion protein had been expressed in turn with at macrophage of spleen, liver, muscle with injection site, and its expression reaches the maximum all of them at immunization144h. It occurred firstly at muscle tissue (immunization24h), the second at spleen (immunization48h), the last at liver (immunization144h). Muscle tissue had sustained more time than spleen and liver, and had more quantity until immunization240h. Liver had the shortest time of continuous expression, and there was no SS fusion protein at immunization240h.
It demonstrated that it was viable to immune livestock the way of intramuscular injection with novel somatostatin DNA vaccine. It was short time that the exogenous plasmid DNA and purpose protein had continuous expression in mice by intramuscular injection.
引文
1.白莉雅.pGM-CSF/SS基因免疫对雌鼠生长和繁殖的影响及其安全性研究.[硕士学位论文].武汉：华中农业大学图书馆,2008
2.白天,王红宁,黄勇,柳萍,周生,胡慧琼,李晓英,唐梦君,潘盛.禽传染性支气管炎DNA疫苗在鸡体内的分布和安全性[J].中国兽医学报,2007,27(5)：628-631
3.曹少先,杨利国,茆达干,张文伟,管峰.生长抑素DNA疫苗pcS/SS质粒的构建及其在HeLa细胞中的表达[J].中国兽医学报,2004,,24(2)：153-156
4.曹少先,杨利国,茆达干,张文伟,邢朝芳.生长抑素DNA疫苗pEGS/2SS在大鼠体内的表达与分布[J].南京农业大学学报,2005,28(2)：137-139
5.曹少先,杨利国,张文伟,茆达干,何晓红.生长抑素基因疫苗质粒pEGS/2SS的构建及表达[J].中国兽医学报,2005,25(5)：499-502
6.曹少先,张文伟,茆达干,管峰,杨利国.生长抑素基因疫苗质粒pcS/2SS的构建、表达及免疫[J].农业生物技术学报,2005,13(4)：477-481
7.曹少先,何晓红,孙延鸣,刘铁铮,杨利国.生长抑素基因疫苗pES/2SS的构建与表达[J].扬州大学学报(农业与生命科学版),2007,28(2)：5-8
8.程宝,基于CpG基序的生长抑素基因新疫苗的构建与鉴定及免疫效果与作用机制的研究.[硕士学位论文].南京：南京农业大学图书馆,2006
9.崔先利,杨利国,茆达干.黄牛对抑制素pCISI基因免疫的反应性[J].吉林农业大学学报,2006,28(2)：197-201
10.戴贤君,方维焕.减毒沙门氏菌为载体的草鱼口服生长抑素DNA疫苗的构建及其稳定性[J].中国兽医学报,2005,25(4)：356-358
11.戴贤君.减毒沙门氏菌为载休的生长抑素ONA疫苗构建、表达及对草鱼生长形响研究.[博士学位论文].杭州：浙江大学图书馆,2005
12.戴建威,章倩倩,王妍,刘松财,张永亮.PLGA微球包裹生长抑素融合基因表达质粒对家兔生长的影响[J].畜牧兽医学报,2008,39(10)：1415-1420
13.邓小玲.DNA疫苗的作用机理以及抗肿瘤的临床应用进展[J].国外医学免疫学分册,2005,28(1)：20—23
14.郭慧琛,孙世琪,刘在新.佐剂及其在基因免疫研究中的应用[J].中国兽医科技,2005,35(4)：315—321
15.郭丽宏,刘建国.DNA疫苗免疫途径的研究现状[J].国外医学病毒学分册,1999,6(1)：9-12
16.侯俊玲,郑亚东,骆学农,才学鹏.DNA疫苗安全性的研究进展[J].细胞与分子免疫学杂志,2009,25(5)：470-472
17.黄维.染色体-质粒平衡致死系统的构建和应用[J].生物技术通讯,2002,13(5)：378-82
18.梁爱心,冯细钢,韩丽,滑国华,桑雷,刘兴斌,刘耘,杨利国.新型生长抑素原核表达质粒的构建及表达鉴定[J].生物工程学报,2008,06,995-998
19.梁爱心.非抗性筛选生长抑素DNA疫苗的构建和免疫效力及安全性研究.[博士学位论文],武汉：华中农业大学图书馆,2009
20.刘建文,乐国伟,施用晖,王志军,汪垣.生长抑素pcDNA3s/SS质粒的构建与鉴定[J]锡轻工大学学报,2002,21(2)：187-190
21.茆达干,张志杰,杨利国,何晓红,张红琳.抑制素基因免疫大鼠的免疫应答与基因表达[J].畜牧兽医学报,2007,38(7)：713-717
22. P.-P. Pastoret, J. blancou,P.Vannier & C.Verschueren编著,才学鹏,景志忠,邱昌庆主译[M],动物疫苗学,北京：中国农业科学出版社,2009
23.舒邓群,茆达干,杨利国,吴志敏,程宝.GM-CSF与生长抑素融合表达质粒对小鼠生长抑素抗体及IgG亚型的影响[J].中国兽医学报,2008,28(1)63-67
24.舒邓群,茆达干,曹少先,杨利国,吴志敏.粒细胞-巨噬细胞集落刺激因子(GM-CSF)与生长抑素(SS)融合表达质粒的构建及其对小鼠的免疫效果[J].农业生物技术学报,2008,16(1)：41-46
25.孙迎基,汪兴生,张新元,沈子龙.生长抑素DNA疫苗在COS细胞中的瞬时表达[J].药物生物技术,2002,9(2)：71-73
26.孙树汉.影响核酸疫苗应用研究的若干问题[J].第二军医大学学报,2002,23(4)：353-354
27.滕家波,苏国富.载体-宿主平衡致死系统在减毒伤寒沙门氏活菌苗中的应用.国外医学免疫学分册[J],1999,22(3)：143-146
28.王云开,曾检华,魏金刚,吴红翔,舒邓群.生长抑素基因疫苗对肉用鸡生长性能及相关激素的影响[J]国兽医学报,2011,31(1)：94—98
29.魏泉德,叶苓,徐劲,余新炳.DNA疫苗在小鼠体内的组织分布及安全性研究[J].中国人兽共患病杂志,2001,17(5)：9-11
30.薛春林,茆达干,杨利国,王勇.生长抑素DNA疫苗对湖羊羔羊生长和相关激素的作用-佐剂效应[J].南京农业大学学报,2007,30(2)：89-93
31.袁正宏,方听,郑玲浩.免疫途径和载体对乙肝病毒DNA疫苗免疫效果影响的研究[J].中华微生物学及免疫学杂志,999,19(1)：25
32.张辉,胡茂志,顾志强,焦新安,耿士忠,张晓明,潘志明.减毒鼠伤寒沙门菌体内定位分析[J].微生物学报,2008,1：48(1)：80-84
33.张剑.奶牛产后生殖机能恢复与生长抑素基因免疫.[博士学位论文],武汉：华中农业大学图书馆,2010
34.张丽娟.堆型艾美耳球虫pcDNA3-3-1E的免疫效应及其安全性研究.[硕士论文],保定：河北农业大学图书馆,2010
35.张志杰.猪卵泡抑制素基因免疫对SD大鼠的安全性研究.[硕士学位论文].南京：南京农业大学图书馆,2008
36.赵兴华,IL-6与GM-CSF基因融合表达生长抑素基因疫苗对小鼠生长的影响.[硕士学位论文],武汉：华中农业大学图书馆,2010
37. Ahmed R K, Biberfeld G and Thorstensson R. Innate immunity in experimental SIV infection and vaccination (J).Molecularlmmunology,2005,42(2):251-258
38. Andre F, Mir LM. DNA electrotransfer:its principles and an updated review of its therapeutic applications. Gene Therapy,2004,1:33-42
39. Anderson RC, Nisbet DJ, Buckley SA, et al. Experimental and natural infection of early weanned pigs with Salmonell acholeraesuis[J]. Res Vet Sci,1998,64 (3): 261-262.
40. Babiuk LA, Pontarollo R, Babiuk S, Loehr B, van Drunen Littel-van den Hurk S. Induction of immune responses by DNA vaccines in large animals.Vaccine,2003, 21(7-8):649-658
41.Bachy M, Boudet M, Gererd-Chambaz Y. Electric pulses increase the immuno genicity of an influenza DNA vaccine injected intramuscularly in the mouse. Vaccine, 2001,19(13-14):1688-1693
42. Barfoed, A. M., B. Kirstensen, et al. (2004). "Influence of routes and administration parameters on antibody response of pigs following DNA vaccination." Vaccine 22(11-12):1395-1405
43. Bauman,D.E.,Eisemann,J.H. And Currie.W.B.,Hormonal effects on partitioning of nutrients for tissue growth:role of growth hormone and prolactin.Federation Proceedings,1982,41:2538-2544
44. Bolton AJ, Osborne MP, Wallis TS, et al. Interaction of Salmonella choleraesuis, Salmonella dublin and Salmonella typhimurium with porcine and bovine terminal ileum in vivo[J]. Microbiology,1999,145(Pt9):2431-2441
45. Bona C, Radu D, Kodera T. Molecular studies on the diversification of hemagglutinin-specific human neonatal repertoire subsequent to immunization with naked DNA. Vaccine,2004,22:1624-30
46. Bot A, Bot S, Garcia-Sastre A, Bona C. DNA immunization of newborn mice with a plasmid-expressing nucleoprotein of influenza virus. Viral Immunol,1996,9(4): 207-210
47. Bouloc A,Walker P,Grivel JC,et al.Immunization through dermal delivery of protein-encoding DNA:a role for migratory dendritic cells[J].Eur J Immunol,1999,29(2):446-454
48. Chen JJ, FangF, LiXZ,etal. Protection against influenza virus infection in BALB/cmice immunized with a single dose of neuramini-dase-expressing DNAs by electroporation [J].Vaccine,2005,23(34):4322-4328
49. Comerota AJ, Throm RC, MillerKA,etal. Naked plasmid DNA encoding fibroblast growth factor type 1 for the treatment of end-stage unreconstructible lower extremity ischemia:Preliminary results of a phase I trial[J].J Vasc Surg,2002,35(5):930-936
50. Darji A, Guzman CA, Gerstel B. Oral somatic transgene vaccination using attenuated S. typhimurium. Cell,1997,91(6):765-775
51. Davis HL, Whalen RG, Demneix BA. Direct gene transfer into skeletal muscle in vivo:Factors affecting efficiency of transfer and stability of expression. Hum Gene Therapy,1993,4(2):151-159
52. Deshmukh TM, Lole KS, Tripathy AS, Arankalle VA. Immunogenicity of candidate hepatitis E virus DNA vaccine expressing complete and truncated ORF2 in mice.Vaccine,2007,25(22):4350-4360
53.Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H,Brose K, Jackson V, Hamada H, Pardoll D and Mulligan RC. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity (J).Proceedings of the National Academy of Sciencesof the USA,1993,90 (8):3539-3543
54. DobanoC, McTagueA, SetteA,etal. Mutating the anchor residues associated with MHC binding inhibits and deviatesCD8+T cell mediated protective immunity against malaria[J].MolImmunol,2007,44(9):2235-2248
55. Elsaesser F, Drath S.The potential of immuno-neutralization of somatostatin for improving pig performance. Livestock Production Science,1995,42(2-3):255-263
56. Ertl HC, Xiang Z. Novel vaccine approaches. J Immunal,1996,156(7):3579-3582
57. Fynan EF, Webster RG, Fuller DH. Haynes JR, Santoro JC, Robinson HL. DNA vaccines:protective immunization by parenteral, mucosal, and gene-gun inoculations. Proc Natl Acad Sci USA,1993,90(24):11478-11482
58. HamersC, JuillardV, FischerL. DNA vaccination against pseudorabies virus and bovine respiratory syncytial virus infections of young animals in the face of maternally derived immunity[J].J CompPathol,2007,137(Suppll):35-41
59. IsaguliantsMG, IakimtchoukK, PetrakovaNV,etal. Gene immunizationmay induce secondary antibodies reacting with DNA[J].Vaccine.2004.22(11-12):1576-1585
60. Klinman D M, Barnhart K M, Co nover J. Cp G motif s as immune adjuvants [J]. Vaccine,1999,17(1):19-25
61.Kwon SS, Kim N, Yoo TJ. The effects of intradermal vaccination with DNA encoding for the T-cell receptoron the induction of experimental autoimmune encephalomyelitis in B10. PL mice[J].JKoreanMed Sci,2005,20(6):1039-1045
62. L.A. Babiuk. R. Pontarollo, S. Babiuk, B. Loehr, S. van Drunen Littel-van den Hurk. Induction of immune responses by DNA vaccines in large animals.Vaccine, 2003,21:649-658
63. Liang AX, Cao SX, Han L, Yao YF, Moaeen-ud-Dina M, Yang LG. Construction and evaluation of the eukaryotic expression plasmid encoding two copies of somatostatin genes fused with hepatitis B surface antigen gene S. Vaccine,2008, 26(23):2935-2941
64. Liang AX, Han L, Hua GH, Geng LY, Sang L, Liu XB, Guo AZ, Yang LG. Construction of a fusion protein expression vector pGS/2SS-M4GFP without antibiotic resistance gene and its subcellular localization in different cell lines. Biologicals,2009,37(1):37-43
65. Ledwith BJ, Manam S, Troilo PJ. Plasmid DNA vaccines:investigation of integration into host cellular DNA following intramuscular injection in mice. Intervirology,2000, 43(4-6):258-272
66. Maeda T, ShintaniY, NakanoK,etal. Failure of inactivated influenza A vaccine to protecthealthy children aged 6-24months[J].Pediatr Int,2004,46(2):122-125
67. Manam S, Ledwith BJ, Barnum AB, Troilo PJ, Pauley CJ, Harper LB, Griffiths TG 2nd, Niu Z, Denisova L, Follmer TT, Pacchione SJ, Wang Z, Beare CM, Bagdon WJ, Nichols WW. Plasmid DNA vaccines:tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA. Intervirology, 2000,43(4-6):273-281
68. Martin T, Parker SE, Hedstrom R, Le T, Hoffman SL, Norman J, Hobart P, Lew D. Plasmid DNA malaria vaccine:the potential for genomic integration after intramuscular injection. Hum Gen Ther,1999,10(5):759-768
69. Meyerholz DK, Stabel TJ. Comparison of early ileal invasionby Salmonella enterica Serovars Choleraesuis and Ty-phimurium[J]. Vet Pathol,2003,40(4):371-375
70. Mor G, Yamshchikov G, Sedegah M. Induction of neonatal tolerance by plasmid DNA vaccination of mice.J Clin Invest,1996,98(12):2700-2705
71.Ou-YangP, HwangLH, TaoMH,et al. Co-delivery ofGM-CSFgene en-hances the immune responses of hepatitis C viral core protein-expressing DNA vaccine:Role of dendritic cells[J].JMed Virol,2002,66:320-328
72. Palka SantiniM, Schwarz-Herzke B, H selM.etal. The gastrointestinal tract as the portal of entry for foreign macromolecules:fate of DNA and proteins[J].Mol Gen Genomics,2003,270(3):201-215
73. Parker SE, Monteith D, Horton H, Hof R, Hernandez P, Vilalta A, Hartikka J, Hobart P, Bentley CE, Chang A, Hedstrom R, Rogers WO, Kumar S, Hoffman SL, Norman JA. Safety of a GM-CSF adjuvant-plasmid DNA malaria vaccine.Gene Ther,2001, 8(13):1011-1023
74. Philip R, Liggitt D, Philip M.In vivo gene delivery. J Biol Chem,1993,268 (22): 16087-16090
75. Robertson JS. Safety considerations for nucleic acid vaccines. Vaccine,1994,12(16): 1526-1528
76. Robertson JS, Griffiths E. Assuring the quality, safety, and efficacy of DNA vaccines. Methods Mol Med,2006,127:363-374
77. Sbai H,Schneider J,Hill AV.Role of transfection in the priming of cytotoxic T-cells by DNA-mediated immunization[J].Vaccine,2002,20(25-26):3137-3147
78. SchubbertR, HohlwegU, RenzD,etal. On the fate of food ingestedforeign DNA in mice:chromosomal associations and placental transmission to the fetus[J].MolGen Genet,1998,259(6):569-576
79. SchleissMR, StroupG, PogorzelskiK,et al. Protection against congenital cytomegalovirus (CMV) disease, conferred by a replication-disabled, bacterial artificial chromosome (BAC)-based DNA vaccine[J].Vaccine,2006,24(37-39): 6175-6186
80. Shiau AL, Chen CC, Yo YT, et al. Enhancement of humoral and cellular immune responses by an oral Salmonella choleraesuis vaccine expressing porcine prothymosin [J]. Vaccine,2005,23(48/49):5563-5571
81. Sizemore D R, Branstrom A A, Sadoff J C. Attenuated bacteria as a DNA delivery vehicle for DNA mediated immunization[J]. Vaccine Elsevier Science,1997,15 (8):804-807
82. Spencer GSG and Williamson ED. Increased growth in lambs following auto-immunization against somatostatin. Anim Prod,1981,32:376-382
83. Spencer GSG. Immunoneutralization of somatostatin (SS) and its effect on animal production. Domest Anim Endocrinol,1986,3:55-68
84. Suezanne E. Parker, Flavia Borellini, Martin L. Wenk, Peter Hobart, Stephen L. Hoffman, Richard Hedstrom, Thong Le, and Jon A. Norman. Plasmid DNA Malaria Vaccine:Tissue Distribution and Safety Studies in Mice and Rabbits.[J].Human Gene Therapy,1999,10 (5):741-758
85. Temin H M. Overview of biological effects of addition of DNA moleculars to cells. J Med Virol,1990,31(1):13-17
86. Tsang CH, MirakhurKK, Babiuk LA,et al. OralDNA immunization in the second trimester fetal lamb and secondary immune responses in the neonate[J].Vaccine,2007, 25(50):8469-8479
87. Tsang Cemaine, Babiuk Shawn, van Drunen Littel-van den Hurk Sylvia, Babiuk Lome A, Philip Griebel. A single DNA immunization in combination with electroporation prolongs the primary immune response and maintains immune memory for six months.Vaccine,2007,25(30):5485-5494
88. Wang Z, Troilo PJ, Wang X, Griffiths TG, Pacchione SJ, Barnum AB, Harper LB, Pauley CJ, Niu Z, Denisova L, Follmer TT, Rizzuto G, Ciliberto G, Fattori E, Monica NL, Manam S, Ledwith BJ.Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation. Gene Ther, 2004,11(8):711-721
89. Watkins C, Hopkins J. Harkiss G. ReporterGene Expression in Dendritic Cells after Gene Gun Administration of Plasmid DNA[J]. Vaccine,2005,23(33):424724256
90. Wehrenbreg WB, Brazeau P, Luben R, Ling N, Guillemin R.A noninvasive functional lesion of the hypothalamo-pituitary axis for the study of growth hormone-releasing factor. Neuroendocrinology,1983,36(6):489
91. Wehrenbreg WB, Brazeau P, Luben P, Bohlen P, Guillemin R. Inhibition of the pulsatile Secetion of growth hormone by monoclonal antibodies to the hypothamic growth releasing factor(GRF). Endocrinology,1982,111(6):2147-2148
92. Weiss S. Transfer of eukaryotic expression plasmids tomammalian hosts by attenuated Salmonella spp [J]. Int J Med Microbiol,2003,93(1):95-106
93. Wolff JA, Ludtke JJ, Acsadi G, Williams P, Jani A.Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet,1992,1(6): 363-369
94. Wolff JA,Dowty ME,Jiao S,et al.Expression of naked plasmids by cultured myotubes and entry of plasmids intoTtubules and caveolae of mammalian skeletal muscle[J].J Cell Sci,1992,103(Pt 4):1249-1259
95. X. B. Liu and A. X. Liang and X. G. Feng and A. Z. Guo and C. Y. Ke and S. J. Zhang and L. G. Yang. Oral and intranasal administration of somatostatin DNA vaccine mediated by attenuated Salmonella Enterica Serovar Typhimurium to promote growth of piglets[J],Animal,2011,8 (5):1231-1236
96. Zhu N, Liggitt D, Liu Y, Debs R. Systemic gene expression after intravenous DNA delivery into adult mice.Science,1993,261(5118):209-11
2.白天,王红宁,黄勇,柳萍,周生,胡慧琼,李晓英,唐梦君,潘盛.禽传染性支气管炎DNA疫苗在鸡体内的分布和安全性[J].中国兽医学报,2007,27(5)：628-631
3.曹少先,杨利国,茆达干,张文伟,管峰.生长抑素DNA疫苗pcS/SS质粒的构建及其在HeLa细胞中的表达[J].中国兽医学报,2004,,24(2)：153-156
4.曹少先,杨利国,茆达干,张文伟,邢朝芳.生长抑素DNA疫苗pEGS/2SS在大鼠体内的表达与分布[J].南京农业大学学报,2005,28(2)：137-139
5.曹少先,杨利国,张文伟,茆达干,何晓红.生长抑素基因疫苗质粒pEGS/2SS的构建及表达[J].中国兽医学报,2005,25(5)：499-502
6.曹少先,张文伟,茆达干,管峰,杨利国.生长抑素基因疫苗质粒pcS/2SS的构建、表达及免疫[J].农业生物技术学报,2005,13(4)：477-481
7.曹少先,何晓红,孙延鸣,刘铁铮,杨利国.生长抑素基因疫苗pES/2SS的构建与表达[J].扬州大学学报(农业与生命科学版),2007,28(2)：5-8
8.程宝,基于CpG基序的生长抑素基因新疫苗的构建与鉴定及免疫效果与作用机制的研究.[硕士学位论文].南京：南京农业大学图书馆,2006
9.崔先利,杨利国,茆达干.黄牛对抑制素pCISI基因免疫的反应性[J].吉林农业大学学报,2006,28(2)：197-201
10.戴贤君,方维焕.减毒沙门氏菌为载体的草鱼口服生长抑素DNA疫苗的构建及其稳定性[J].中国兽医学报,2005,25(4)：356-358
11.戴贤君.减毒沙门氏菌为载休的生长抑素ONA疫苗构建、表达及对草鱼生长形响研究.[博士学位论文].杭州：浙江大学图书馆,2005
12.戴建威,章倩倩,王妍,刘松财,张永亮.PLGA微球包裹生长抑素融合基因表达质粒对家兔生长的影响[J].畜牧兽医学报,2008,39(10)：1415-1420
13.邓小玲.DNA疫苗的作用机理以及抗肿瘤的临床应用进展[J].国外医学免疫学分册,2005,28(1)：20—23
14.郭慧琛,孙世琪,刘在新.佐剂及其在基因免疫研究中的应用[J].中国兽医科技,2005,35(4)：315—321
15.郭丽宏,刘建国.DNA疫苗免疫途径的研究现状[J].国外医学病毒学分册,1999,6(1)：9-12
16.侯俊玲,郑亚东,骆学农,才学鹏.DNA疫苗安全性的研究进展[J].细胞与分子免疫学杂志,2009,25(5)：470-472
17.黄维.染色体-质粒平衡致死系统的构建和应用[J].生物技术通讯,2002,13(5)：378-82
18.梁爱心,冯细钢,韩丽,滑国华,桑雷,刘兴斌,刘耘,杨利国.新型生长抑素原核表达质粒的构建及表达鉴定[J].生物工程学报,2008,06,995-998
19.梁爱心.非抗性筛选生长抑素DNA疫苗的构建和免疫效力及安全性研究.[博士学位论文],武汉：华中农业大学图书馆,2009
20.刘建文,乐国伟,施用晖,王志军,汪垣.生长抑素pcDNA3s/SS质粒的构建与鉴定[J]锡轻工大学学报,2002,21(2)：187-190
21.茆达干,张志杰,杨利国,何晓红,张红琳.抑制素基因免疫大鼠的免疫应答与基因表达[J].畜牧兽医学报,2007,38(7)：713-717
22. P.-P. Pastoret, J. blancou,P.Vannier & C.Verschueren编著,才学鹏,景志忠,邱昌庆主译[M],动物疫苗学,北京：中国农业科学出版社,2009
23.舒邓群,茆达干,杨利国,吴志敏,程宝.GM-CSF与生长抑素融合表达质粒对小鼠生长抑素抗体及IgG亚型的影响[J].中国兽医学报,2008,28(1)63-67
24.舒邓群,茆达干,曹少先,杨利国,吴志敏.粒细胞-巨噬细胞集落刺激因子(GM-CSF)与生长抑素(SS)融合表达质粒的构建及其对小鼠的免疫效果[J].农业生物技术学报,2008,16(1)：41-46
25.孙迎基,汪兴生,张新元,沈子龙.生长抑素DNA疫苗在COS细胞中的瞬时表达[J].药物生物技术,2002,9(2)：71-73
26.孙树汉.影响核酸疫苗应用研究的若干问题[J].第二军医大学学报,2002,23(4)：353-354
27.滕家波,苏国富.载体-宿主平衡致死系统在减毒伤寒沙门氏活菌苗中的应用.国外医学免疫学分册[J],1999,22(3)：143-146
28.王云开,曾检华,魏金刚,吴红翔,舒邓群.生长抑素基因疫苗对肉用鸡生长性能及相关激素的影响[J]国兽医学报,2011,31(1)：94—98
29.魏泉德,叶苓,徐劲,余新炳.DNA疫苗在小鼠体内的组织分布及安全性研究[J].中国人兽共患病杂志,2001,17(5)：9-11
30.薛春林,茆达干,杨利国,王勇.生长抑素DNA疫苗对湖羊羔羊生长和相关激素的作用-佐剂效应[J].南京农业大学学报,2007,30(2)：89-93
31.袁正宏,方听,郑玲浩.免疫途径和载体对乙肝病毒DNA疫苗免疫效果影响的研究[J].中华微生物学及免疫学杂志,999,19(1)：25
32.张辉,胡茂志,顾志强,焦新安,耿士忠,张晓明,潘志明.减毒鼠伤寒沙门菌体内定位分析[J].微生物学报,2008,1：48(1)：80-84
33.张剑.奶牛产后生殖机能恢复与生长抑素基因免疫.[博士学位论文],武汉：华中农业大学图书馆,2010
34.张丽娟.堆型艾美耳球虫pcDNA3-3-1E的免疫效应及其安全性研究.[硕士论文],保定：河北农业大学图书馆,2010
35.张志杰.猪卵泡抑制素基因免疫对SD大鼠的安全性研究.[硕士学位论文].南京：南京农业大学图书馆,2008
36.赵兴华,IL-6与GM-CSF基因融合表达生长抑素基因疫苗对小鼠生长的影响.[硕士学位论文],武汉：华中农业大学图书馆,2010
37. Ahmed R K, Biberfeld G and Thorstensson R. Innate immunity in experimental SIV infection and vaccination (J).Molecularlmmunology,2005,42(2):251-258
38. Andre F, Mir LM. DNA electrotransfer:its principles and an updated review of its therapeutic applications. Gene Therapy,2004,1:33-42
39. Anderson RC, Nisbet DJ, Buckley SA, et al. Experimental and natural infection of early weanned pigs with Salmonell acholeraesuis[J]. Res Vet Sci,1998,64 (3): 261-262.
40. Babiuk LA, Pontarollo R, Babiuk S, Loehr B, van Drunen Littel-van den Hurk S. Induction of immune responses by DNA vaccines in large animals.Vaccine,2003, 21(7-8):649-658
41.Bachy M, Boudet M, Gererd-Chambaz Y. Electric pulses increase the immuno genicity of an influenza DNA vaccine injected intramuscularly in the mouse. Vaccine, 2001,19(13-14):1688-1693
42. Barfoed, A. M., B. Kirstensen, et al. (2004). "Influence of routes and administration parameters on antibody response of pigs following DNA vaccination." Vaccine 22(11-12):1395-1405
43. Bauman,D.E.,Eisemann,J.H. And Currie.W.B.,Hormonal effects on partitioning of nutrients for tissue growth:role of growth hormone and prolactin.Federation Proceedings,1982,41:2538-2544
44. Bolton AJ, Osborne MP, Wallis TS, et al. Interaction of Salmonella choleraesuis, Salmonella dublin and Salmonella typhimurium with porcine and bovine terminal ileum in vivo[J]. Microbiology,1999,145(Pt9):2431-2441
45. Bona C, Radu D, Kodera T. Molecular studies on the diversification of hemagglutinin-specific human neonatal repertoire subsequent to immunization with naked DNA. Vaccine,2004,22:1624-30
46. Bot A, Bot S, Garcia-Sastre A, Bona C. DNA immunization of newborn mice with a plasmid-expressing nucleoprotein of influenza virus. Viral Immunol,1996,9(4): 207-210
47. Bouloc A,Walker P,Grivel JC,et al.Immunization through dermal delivery of protein-encoding DNA:a role for migratory dendritic cells[J].Eur J Immunol,1999,29(2):446-454
48. Chen JJ, FangF, LiXZ,etal. Protection against influenza virus infection in BALB/cmice immunized with a single dose of neuramini-dase-expressing DNAs by electroporation [J].Vaccine,2005,23(34):4322-4328
49. Comerota AJ, Throm RC, MillerKA,etal. Naked plasmid DNA encoding fibroblast growth factor type 1 for the treatment of end-stage unreconstructible lower extremity ischemia:Preliminary results of a phase I trial[J].J Vasc Surg,2002,35(5):930-936
50. Darji A, Guzman CA, Gerstel B. Oral somatic transgene vaccination using attenuated S. typhimurium. Cell,1997,91(6):765-775
51. Davis HL, Whalen RG, Demneix BA. Direct gene transfer into skeletal muscle in vivo:Factors affecting efficiency of transfer and stability of expression. Hum Gene Therapy,1993,4(2):151-159
52. Deshmukh TM, Lole KS, Tripathy AS, Arankalle VA. Immunogenicity of candidate hepatitis E virus DNA vaccine expressing complete and truncated ORF2 in mice.Vaccine,2007,25(22):4350-4360
53.Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H,Brose K, Jackson V, Hamada H, Pardoll D and Mulligan RC. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity (J).Proceedings of the National Academy of Sciencesof the USA,1993,90 (8):3539-3543
54. DobanoC, McTagueA, SetteA,etal. Mutating the anchor residues associated with MHC binding inhibits and deviatesCD8+T cell mediated protective immunity against malaria[J].MolImmunol,2007,44(9):2235-2248
55. Elsaesser F, Drath S.The potential of immuno-neutralization of somatostatin for improving pig performance. Livestock Production Science,1995,42(2-3):255-263
56. Ertl HC, Xiang Z. Novel vaccine approaches. J Immunal,1996,156(7):3579-3582
57. Fynan EF, Webster RG, Fuller DH. Haynes JR, Santoro JC, Robinson HL. DNA vaccines:protective immunization by parenteral, mucosal, and gene-gun inoculations. Proc Natl Acad Sci USA,1993,90(24):11478-11482
58. HamersC, JuillardV, FischerL. DNA vaccination against pseudorabies virus and bovine respiratory syncytial virus infections of young animals in the face of maternally derived immunity[J].J CompPathol,2007,137(Suppll):35-41
59. IsaguliantsMG, IakimtchoukK, PetrakovaNV,etal. Gene immunizationmay induce secondary antibodies reacting with DNA[J].Vaccine.2004.22(11-12):1576-1585
60. Klinman D M, Barnhart K M, Co nover J. Cp G motif s as immune adjuvants [J]. Vaccine,1999,17(1):19-25
61.Kwon SS, Kim N, Yoo TJ. The effects of intradermal vaccination with DNA encoding for the T-cell receptoron the induction of experimental autoimmune encephalomyelitis in B10. PL mice[J].JKoreanMed Sci,2005,20(6):1039-1045
62. L.A. Babiuk. R. Pontarollo, S. Babiuk, B. Loehr, S. van Drunen Littel-van den Hurk. Induction of immune responses by DNA vaccines in large animals.Vaccine, 2003,21:649-658
63. Liang AX, Cao SX, Han L, Yao YF, Moaeen-ud-Dina M, Yang LG. Construction and evaluation of the eukaryotic expression plasmid encoding two copies of somatostatin genes fused with hepatitis B surface antigen gene S. Vaccine,2008, 26(23):2935-2941
64. Liang AX, Han L, Hua GH, Geng LY, Sang L, Liu XB, Guo AZ, Yang LG. Construction of a fusion protein expression vector pGS/2SS-M4GFP without antibiotic resistance gene and its subcellular localization in different cell lines. Biologicals,2009,37(1):37-43
65. Ledwith BJ, Manam S, Troilo PJ. Plasmid DNA vaccines:investigation of integration into host cellular DNA following intramuscular injection in mice. Intervirology,2000, 43(4-6):258-272
66. Maeda T, ShintaniY, NakanoK,etal. Failure of inactivated influenza A vaccine to protecthealthy children aged 6-24months[J].Pediatr Int,2004,46(2):122-125
67. Manam S, Ledwith BJ, Barnum AB, Troilo PJ, Pauley CJ, Harper LB, Griffiths TG 2nd, Niu Z, Denisova L, Follmer TT, Pacchione SJ, Wang Z, Beare CM, Bagdon WJ, Nichols WW. Plasmid DNA vaccines:tissue distribution and effects of DNA sequence, adjuvants and delivery method on integration into host DNA. Intervirology, 2000,43(4-6):273-281
68. Martin T, Parker SE, Hedstrom R, Le T, Hoffman SL, Norman J, Hobart P, Lew D. Plasmid DNA malaria vaccine:the potential for genomic integration after intramuscular injection. Hum Gen Ther,1999,10(5):759-768
69. Meyerholz DK, Stabel TJ. Comparison of early ileal invasionby Salmonella enterica Serovars Choleraesuis and Ty-phimurium[J]. Vet Pathol,2003,40(4):371-375
70. Mor G, Yamshchikov G, Sedegah M. Induction of neonatal tolerance by plasmid DNA vaccination of mice.J Clin Invest,1996,98(12):2700-2705
71.Ou-YangP, HwangLH, TaoMH,et al. Co-delivery ofGM-CSFgene en-hances the immune responses of hepatitis C viral core protein-expressing DNA vaccine:Role of dendritic cells[J].JMed Virol,2002,66:320-328
72. Palka SantiniM, Schwarz-Herzke B, H selM.etal. The gastrointestinal tract as the portal of entry for foreign macromolecules:fate of DNA and proteins[J].Mol Gen Genomics,2003,270(3):201-215
73. Parker SE, Monteith D, Horton H, Hof R, Hernandez P, Vilalta A, Hartikka J, Hobart P, Bentley CE, Chang A, Hedstrom R, Rogers WO, Kumar S, Hoffman SL, Norman JA. Safety of a GM-CSF adjuvant-plasmid DNA malaria vaccine.Gene Ther,2001, 8(13):1011-1023
74. Philip R, Liggitt D, Philip M.In vivo gene delivery. J Biol Chem,1993,268 (22): 16087-16090
75. Robertson JS. Safety considerations for nucleic acid vaccines. Vaccine,1994,12(16): 1526-1528
76. Robertson JS, Griffiths E. Assuring the quality, safety, and efficacy of DNA vaccines. Methods Mol Med,2006,127:363-374
77. Sbai H,Schneider J,Hill AV.Role of transfection in the priming of cytotoxic T-cells by DNA-mediated immunization[J].Vaccine,2002,20(25-26):3137-3147
78. SchubbertR, HohlwegU, RenzD,etal. On the fate of food ingestedforeign DNA in mice:chromosomal associations and placental transmission to the fetus[J].MolGen Genet,1998,259(6):569-576
79. SchleissMR, StroupG, PogorzelskiK,et al. Protection against congenital cytomegalovirus (CMV) disease, conferred by a replication-disabled, bacterial artificial chromosome (BAC)-based DNA vaccine[J].Vaccine,2006,24(37-39): 6175-6186
80. Shiau AL, Chen CC, Yo YT, et al. Enhancement of humoral and cellular immune responses by an oral Salmonella choleraesuis vaccine expressing porcine prothymosin [J]. Vaccine,2005,23(48/49):5563-5571
81. Sizemore D R, Branstrom A A, Sadoff J C. Attenuated bacteria as a DNA delivery vehicle for DNA mediated immunization[J]. Vaccine Elsevier Science,1997,15 (8):804-807
82. Spencer GSG and Williamson ED. Increased growth in lambs following auto-immunization against somatostatin. Anim Prod,1981,32:376-382
83. Spencer GSG. Immunoneutralization of somatostatin (SS) and its effect on animal production. Domest Anim Endocrinol,1986,3:55-68
84. Suezanne E. Parker, Flavia Borellini, Martin L. Wenk, Peter Hobart, Stephen L. Hoffman, Richard Hedstrom, Thong Le, and Jon A. Norman. Plasmid DNA Malaria Vaccine:Tissue Distribution and Safety Studies in Mice and Rabbits.[J].Human Gene Therapy,1999,10 (5):741-758
85. Temin H M. Overview of biological effects of addition of DNA moleculars to cells. J Med Virol,1990,31(1):13-17
86. Tsang CH, MirakhurKK, Babiuk LA,et al. OralDNA immunization in the second trimester fetal lamb and secondary immune responses in the neonate[J].Vaccine,2007, 25(50):8469-8479
87. Tsang Cemaine, Babiuk Shawn, van Drunen Littel-van den Hurk Sylvia, Babiuk Lome A, Philip Griebel. A single DNA immunization in combination with electroporation prolongs the primary immune response and maintains immune memory for six months.Vaccine,2007,25(30):5485-5494
88. Wang Z, Troilo PJ, Wang X, Griffiths TG, Pacchione SJ, Barnum AB, Harper LB, Pauley CJ, Niu Z, Denisova L, Follmer TT, Rizzuto G, Ciliberto G, Fattori E, Monica NL, Manam S, Ledwith BJ.Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation. Gene Ther, 2004,11(8):711-721
89. Watkins C, Hopkins J. Harkiss G. ReporterGene Expression in Dendritic Cells after Gene Gun Administration of Plasmid DNA[J]. Vaccine,2005,23(33):424724256
90. Wehrenbreg WB, Brazeau P, Luben R, Ling N, Guillemin R.A noninvasive functional lesion of the hypothalamo-pituitary axis for the study of growth hormone-releasing factor. Neuroendocrinology,1983,36(6):489
91. Wehrenbreg WB, Brazeau P, Luben P, Bohlen P, Guillemin R. Inhibition of the pulsatile Secetion of growth hormone by monoclonal antibodies to the hypothamic growth releasing factor(GRF). Endocrinology,1982,111(6):2147-2148
92. Weiss S. Transfer of eukaryotic expression plasmids tomammalian hosts by attenuated Salmonella spp [J]. Int J Med Microbiol,2003,93(1):95-106
93. Wolff JA, Ludtke JJ, Acsadi G, Williams P, Jani A.Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet,1992,1(6): 363-369
94. Wolff JA,Dowty ME,Jiao S,et al.Expression of naked plasmids by cultured myotubes and entry of plasmids intoTtubules and caveolae of mammalian skeletal muscle[J].J Cell Sci,1992,103(Pt 4):1249-1259
95. X. B. Liu and A. X. Liang and X. G. Feng and A. Z. Guo and C. Y. Ke and S. J. Zhang and L. G. Yang. Oral and intranasal administration of somatostatin DNA vaccine mediated by attenuated Salmonella Enterica Serovar Typhimurium to promote growth of piglets[J],Animal,2011,8 (5):1231-1236
96. Zhu N, Liggitt D, Liu Y, Debs R. Systemic gene expression after intravenous DNA delivery into adult mice.Science,1993,261(5118):209-11