猪瘟伪狂犬病重组病毒活疫苗的研究
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摘要
试验中采用直接荧光抗体检测、兔体交互免疫试验和ELISA检测方法确诊猪瘟临床病例，分离了猪瘟野毒MZ株。采用RT-PCR扩增了MZ株的囊膜糖蛋白E2基因全序列，克隆于质粒PUC18中并进行了核苷酸序列测定及氨基酸序列推导。将其与猪瘟病毒(HCV)石门株(SM)株和Brescia株对应序列进行了同源性比较，结果核苷酸同源性为95％和94％，氨基酸为96％和94.2％。克隆片段长1285bp包含HCV E2基因全序列，编码包含信号肽及跨膜区在内的完整囊膜糖蛋白E2氨基酸序列。对该氨基酸序列进行疏水性及氨基酸变异分析，未见其抗原决定簇发生明显变异。
为了构建猪瘟伪狂犬病重组病毒，我们采用BamHI和HindIII双酶切重组质粒PhcvE2获得E2基因片段，与经BclI和EcoRV双酶切处理的PP63LacZ质粒DNA连接转化大肠杆菌DH5a，构建了转移重组质粒PP63LacZE2。经BclI和SacI双酶切鉴定证明该重组质粒PP63LacZE2含有HCV E2基因。采用磷酸钙转染系统，将伪狂犬病三基因缺失疫苗SA215株DNA与PP63LacZE2 DNA共转染Vero细胞，获得SA215(A)1、SA215(A)2和SA215(A)3等12个经同源重组后在含X-gal覆盖琼脂中产生的白色病毒噬斑。以光生物素标记的HCV E2基因为探针进行初步鉴定，结果均为阳性。挑选SA215(A)1株作BamHI酶切和southern转印杂交鉴定，结果表明重组病毒构建是成功的，将其命名为SA215(A)。直接荧光抗体检测、病毒蛋白质SDS-PAGE电泳和western免疫印迹检测结果表明HCV E2基因在重组病毒内获得表达，产生大小为51Kd左右囊膜糖蛋，白E2。对重组病毒SA215(A)株进行部分生物学特性研究，经过培养特性观察试验表明该毒株可适应Vero细胞、BHK21细胞、IBRS细胞、PK15细胞、MDBK细胞和鸡胚成纤维细胞，但对不同细胞系表现有一定的差异。比较发现其与SA215株致细胞病变能力存在很大差异，在Vero细胞上重组病毒SA215(A)株引起细胞病变所需时间延长，且最终形成的空斑大大小于亲本病毒SA215。重组病毒SA215(A)株能被特异性抗伪狂犬病病毒血清完全中和，其仍具有亲本病毒血清学特性。电镜观察未发现重组病毒SA215(A)与亲本病毒之间存在形
态学差异，但可见其增殖能力明显降低。核酸杂交检测结果表明，SAZ巧(A)
株与SA215株在动物体内分布情况一致，仅能定植于三叉神经节以下。SAZ巧
(A)株lml接种21日龄健康仔猪(伪狂犬病和猪瘟检测阴性)，在接种后28
天用10毕FU的PRV Fa强毒株进行滴鼻攻毒，42天用猪瘟强毒SM株血毒lml
肌肉注射攻击，结果表明接种猪能抵御两次病毒攻击，而同条件下对照结果成
立，表明SAZ 15(A)株具有良好免疫原性。以5 x 10‘、1 x 10，、5 x 1 05和1 x 1 06PFU
等不同剂量的SAZ15(A)株接种21日龄健康仔猪(伪狂犬病和猪瘟检测阴性)，
按上述条件进行PRv和HCv攻毒试验确定其最低免疫剂量为1 x 1 osPFU。
以重组病毒SAZ 15(A)株为种毒，我们试定了猪瘟伪狂犬病重组病毒活疫
苗的生产检验工艺，选择鸡胚成纤维细胞为生产用细胞，vero细胞为检验用细
胞，试制了01001、01002和01003等3批猪瘟伪狂犬病重组病毒活疫苗。对每
批疫苗半成品和成品进行抽样检验，结果3批疫苗均为合格产品。试制的3批
疫苗物理性状检验合格，无细菌、霉菌、支原体和外源病毒污染;剩余水分分
别为2.6%、2.7%和2.3%:疫苗真空度检测结果均为ZPa:批间毒价稳定，每头
份疫苗病毒含量均在1护PFU以上。根据保存期试验所得结果:猪瘟伪狂犬病重
组病毒活疫苗SA215(A)在一200C保存1年未引起毒价下降;4一80C保存1年毒
价仅轻微下降，但仍然满足疫苗最低免疫剂量要求;室温条件下保存时短期内
即表现毒价明显下降，故将疫苗SAZ15(A)保存期确定为4一80C保存1年。安
全性试验结果表明不同剂量的疫苗SAZ15(A)接种经检测为伪狂犬病和猪瘟阴
性的新生仔猪、21日龄健康仔猪及妊娠母猪均未见异常反应，证明了该疫苗的
安全性。通过在接种疫苗后不同时间内对动物进行血清抗体检测或攻毒后保护
率计算，结果表明测定期内疫苗SAZ15(A)接种动物均可抵御PRV和HCV强
毒攻击，结合伪狂犬病发病特点将疫苗的免疫期定为28周。上述试验结果说明，
重组病毒SAZ15(A)是一株优秀的猪瘟、伪狂犬病二价疫苗候选株。
By means of IFT、 rabbit cross immunity test and ELISA we diagnosed the clinical case of Hog Cholera Virus (HCV).The MZ strain of HCV was isolated. Full-length fragments of envelope glycoprotein E2gene of MZ strain amplified with RT-PCR was cloned into PUC18 easy vector. The result showed that HCV E2 gene of MZ strain share 95%,94% with HCV SM strain, Brescia strain in nucleotide sequence respectively, and share 96%,94.2% identity in amino acids sequence respectively. A 1285bp cloned fragments encoding intact envelope glycoprotein E2 was obtained, including its signal peptide sequence (WLLLVTGA) and transmenbrane region (TMR).This study showed epitope was not variable obviously by analysising the hydrophobicity and the varintion of amino acid.It could be high immunogenicity.
To construct recombinant virus of PRV and HCV,E2 gene fragment from recombinant plasmid digested with BamHI and Hindlll was cloned in the plasmid PP63LacZ which digested with Bell and EcoRV and transformed into strain DH5 a of E.coli, yielding plasmid PP63LacZE2.The recombinant plasmid PP63LacZE2 was indicated containing E2 gene by PCR and Dot-blot.By cotransfection of PRV Fa TK/gE-/gI-/LacZ (SA215) DNA and recombinant plasmid PP63LacZE2 DNA in Vero cells, 12 recombinant namly SA215(A)1 , SA215(A)2 et al were obtained with calcium phosphate transfection system.The results obtained from biotinlabeled probe of HCV E2,indicating positive. Through digestion with BamHI and Southern-blot acid hybridization of SA215(A)1 strain, this study showed the recombinant virus of PRV and HCV namly SA215(A) was successful. IFT, SDS-PAGE, electrophoresis and Western-blot confirmed HCV E2 gene was expressed in recombinant virus, about 51Kd fragment of envelope glycoprotein E2 gene. This study on the biological characteristics o
f SA215(A) strain showed this strain could replicated very well on vero cells, BHK21 cells et al. There were variability of cell culture at different cell lines. There was a great variation in inducing CPE between SA215 strain and SA215(A) strain: the time of SA215(A) inducing CPE on vero cell was prolonged, final plaque was smaller than parent virus SA215. SA215(A) was
neutralized by positive sera of PRV, SA215(A) was of serum properties of parent virus . No-morphdogical difference between SA215 and SA215(A) and obviously fall of reproductive capacity of SA215(A) under electron microscope. Through DOT-blot hybridiztion the result indicated SA215(A) and SA215 were of the same distribution on animal ,simply planting the following trigeminal ganglion .21-day-old SPF pigs inoculated intramuscularly SA215(A) strain could be protected from being affected at day 28 after oronasal challenge-exposure with 106 PF.U high-dose virulent PRV Fa strain and at day 42 after i.m. challenge-exposure with 1 ml high-dose virulent HCV SM strain ,when control groups were tenable ,the result showed SA215(A) was high efficacy .2121-day-old SPF pigs vaccinated i.m. with 5 *104, 1* 10s, 5 * 105, 1* 106P.F.U. SA215(A) strain ,challenge-exposure under the same conditions. The lowest immune dosage was determined 1*105 P.F.U.
We made the production check technology were of recombinant live vaccine of PRV and HCV,SA215(A) strain was used as virus stock .production and test select chicken embryonic fibre cell and vero cell, repecticly three lot of live vaccine were prepared (01001,01002 and 01003) .The result indicated three lot of vaccines were up to standard . The titer of the vaccine was stable at different lot. The titer of a field dose was usually #105P.F.U. It was not significantly reduced after keeping the vaccine at -20@ and 4-8 @ for one year, but it was obviously reduced at room temperature for short-term, therefore the length of protection was determined at 4-8@ for one year. The safety was tested in newborn piglets,21-day-old post-weaning piglets and pregnant sows injected different dosage of.The fact that no any reverse reaction indicated the high safety of the vaccine. Through titers of antibodies test from injected animal of vaccine in d
为了构建猪瘟伪狂犬病重组病毒，我们采用BamHI和HindIII双酶切重组质粒PhcvE2获得E2基因片段，与经BclI和EcoRV双酶切处理的PP63LacZ质粒DNA连接转化大肠杆菌DH5a，构建了转移重组质粒PP63LacZE2。经BclI和SacI双酶切鉴定证明该重组质粒PP63LacZE2含有HCV E2基因。采用磷酸钙转染系统，将伪狂犬病三基因缺失疫苗SA215株DNA与PP63LacZE2 DNA共转染Vero细胞，获得SA215(A)1、SA215(A)2和SA215(A)3等12个经同源重组后在含X-gal覆盖琼脂中产生的白色病毒噬斑。以光生物素标记的HCV E2基因为探针进行初步鉴定，结果均为阳性。挑选SA215(A)1株作BamHI酶切和southern转印杂交鉴定，结果表明重组病毒构建是成功的，将其命名为SA215(A)。直接荧光抗体检测、病毒蛋白质SDS-PAGE电泳和western免疫印迹检测结果表明HCV E2基因在重组病毒内获得表达，产生大小为51Kd左右囊膜糖蛋，白E2。对重组病毒SA215(A)株进行部分生物学特性研究，经过培养特性观察试验表明该毒株可适应Vero细胞、BHK21细胞、IBRS细胞、PK15细胞、MDBK细胞和鸡胚成纤维细胞，但对不同细胞系表现有一定的差异。比较发现其与SA215株致细胞病变能力存在很大差异，在Vero细胞上重组病毒SA215(A)株引起细胞病变所需时间延长，且最终形成的空斑大大小于亲本病毒SA215。重组病毒SA215(A)株能被特异性抗伪狂犬病病毒血清完全中和，其仍具有亲本病毒血清学特性。电镜观察未发现重组病毒SA215(A)与亲本病毒之间存在形
态学差异，但可见其增殖能力明显降低。核酸杂交检测结果表明，SAZ巧(A)
株与SA215株在动物体内分布情况一致，仅能定植于三叉神经节以下。SAZ巧
(A)株lml接种21日龄健康仔猪(伪狂犬病和猪瘟检测阴性)，在接种后28
天用10毕FU的PRV Fa强毒株进行滴鼻攻毒，42天用猪瘟强毒SM株血毒lml
肌肉注射攻击，结果表明接种猪能抵御两次病毒攻击，而同条件下对照结果成
立，表明SAZ 15(A)株具有良好免疫原性。以5 x 10‘、1 x 10，、5 x 1 05和1 x 1 06PFU
等不同剂量的SAZ15(A)株接种21日龄健康仔猪(伪狂犬病和猪瘟检测阴性)，
按上述条件进行PRv和HCv攻毒试验确定其最低免疫剂量为1 x 1 osPFU。
以重组病毒SAZ 15(A)株为种毒，我们试定了猪瘟伪狂犬病重组病毒活疫
苗的生产检验工艺，选择鸡胚成纤维细胞为生产用细胞，vero细胞为检验用细
胞，试制了01001、01002和01003等3批猪瘟伪狂犬病重组病毒活疫苗。对每
批疫苗半成品和成品进行抽样检验，结果3批疫苗均为合格产品。试制的3批
疫苗物理性状检验合格，无细菌、霉菌、支原体和外源病毒污染;剩余水分分
别为2.6%、2.7%和2.3%:疫苗真空度检测结果均为ZPa:批间毒价稳定，每头
份疫苗病毒含量均在1护PFU以上。根据保存期试验所得结果:猪瘟伪狂犬病重
组病毒活疫苗SA215(A)在一200C保存1年未引起毒价下降;4一80C保存1年毒
价仅轻微下降，但仍然满足疫苗最低免疫剂量要求;室温条件下保存时短期内
即表现毒价明显下降，故将疫苗SAZ15(A)保存期确定为4一80C保存1年。安
全性试验结果表明不同剂量的疫苗SAZ15(A)接种经检测为伪狂犬病和猪瘟阴
性的新生仔猪、21日龄健康仔猪及妊娠母猪均未见异常反应，证明了该疫苗的
安全性。通过在接种疫苗后不同时间内对动物进行血清抗体检测或攻毒后保护
率计算，结果表明测定期内疫苗SAZ15(A)接种动物均可抵御PRV和HCV强
毒攻击，结合伪狂犬病发病特点将疫苗的免疫期定为28周。上述试验结果说明，
重组病毒SAZ15(A)是一株优秀的猪瘟、伪狂犬病二价疫苗候选株。
By means of IFT、 rabbit cross immunity test and ELISA we diagnosed the clinical case of Hog Cholera Virus (HCV).The MZ strain of HCV was isolated. Full-length fragments of envelope glycoprotein E2gene of MZ strain amplified with RT-PCR was cloned into PUC18 easy vector. The result showed that HCV E2 gene of MZ strain share 95%,94% with HCV SM strain, Brescia strain in nucleotide sequence respectively, and share 96%,94.2% identity in amino acids sequence respectively. A 1285bp cloned fragments encoding intact envelope glycoprotein E2 was obtained, including its signal peptide sequence (WLLLVTGA) and transmenbrane region (TMR).This study showed epitope was not variable obviously by analysising the hydrophobicity and the varintion of amino acid.It could be high immunogenicity.
To construct recombinant virus of PRV and HCV,E2 gene fragment from recombinant plasmid digested with BamHI and Hindlll was cloned in the plasmid PP63LacZ which digested with Bell and EcoRV and transformed into strain DH5 a of E.coli, yielding plasmid PP63LacZE2.The recombinant plasmid PP63LacZE2 was indicated containing E2 gene by PCR and Dot-blot.By cotransfection of PRV Fa TK/gE-/gI-/LacZ (SA215) DNA and recombinant plasmid PP63LacZE2 DNA in Vero cells, 12 recombinant namly SA215(A)1 , SA215(A)2 et al were obtained with calcium phosphate transfection system.The results obtained from biotinlabeled probe of HCV E2,indicating positive. Through digestion with BamHI and Southern-blot acid hybridization of SA215(A)1 strain, this study showed the recombinant virus of PRV and HCV namly SA215(A) was successful. IFT, SDS-PAGE, electrophoresis and Western-blot confirmed HCV E2 gene was expressed in recombinant virus, about 51Kd fragment of envelope glycoprotein E2 gene. This study on the biological characteristics o
f SA215(A) strain showed this strain could replicated very well on vero cells, BHK21 cells et al. There were variability of cell culture at different cell lines. There was a great variation in inducing CPE between SA215 strain and SA215(A) strain: the time of SA215(A) inducing CPE on vero cell was prolonged, final plaque was smaller than parent virus SA215. SA215(A) was
neutralized by positive sera of PRV, SA215(A) was of serum properties of parent virus . No-morphdogical difference between SA215 and SA215(A) and obviously fall of reproductive capacity of SA215(A) under electron microscope. Through DOT-blot hybridiztion the result indicated SA215(A) and SA215 were of the same distribution on animal ,simply planting the following trigeminal ganglion .21-day-old SPF pigs inoculated intramuscularly SA215(A) strain could be protected from being affected at day 28 after oronasal challenge-exposure with 106 PF.U high-dose virulent PRV Fa strain and at day 42 after i.m. challenge-exposure with 1 ml high-dose virulent HCV SM strain ,when control groups were tenable ,the result showed SA215(A) was high efficacy .2121-day-old SPF pigs vaccinated i.m. with 5 *104, 1* 10s, 5 * 105, 1* 106P.F.U. SA215(A) strain ,challenge-exposure under the same conditions. The lowest immune dosage was determined 1*105 P.F.U.
We made the production check technology were of recombinant live vaccine of PRV and HCV,SA215(A) strain was used as virus stock .production and test select chicken embryonic fibre cell and vero cell, repecticly three lot of live vaccine were prepared (01001,01002 and 01003) .The result indicated three lot of vaccines were up to standard . The titer of the vaccine was stable at different lot. The titer of a field dose was usually #105P.F.U. It was not significantly reduced after keeping the vaccine at -20@ and 4-8 @ for one year, but it was obviously reduced at room temperature for short-term, therefore the length of protection was determined at 4-8@ for one year. The safety was tested in newborn piglets,21-day-old post-weaning piglets and pregnant sows injected different dosage of.The fact that no any reverse reaction indicated the high safety of the vaccine. Through titers of antibodies test from injected animal of vaccine in d
引文
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