猪伪狂犬病病毒豫A株的分离、鉴定及PK基因的克隆与序列分析
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摘要
从河南某猪场发生疑似伪狂犬病（Pr）的仔猪体内无菌采取病料（脑、淋巴结、三叉神经、肝脏和肾脏等组织），研磨，离心，上清液加入青、链霉素至终浓度为1000u/ml，接种长成单层的PK15细胞，连续传4代，均能产生明显的细胞病变（CPE）；取病料悬液和细胞培养液接种家兔进行动物实验，家兔没有表现出典型的Pr症状，接毒后20天利用猪伪狂犬病乳胶凝集检测试剂盒检测到伪狂犬病病毒（Prv）抗体，滴度达1:4－1:16。参考GeneBank公布的猪伪狂犬病病毒的PK基因序列，自行设计了一对针对Prv PK基因的引物，进行聚合酶链式反应（PCR），在该份病料和第4代细胞培养液中均可检测到预期大小的全长目的基因片段。以上试验证明分离到了河南地方株Prv，命名为豫A（Yu A）株。
利用上述引物，对来自河南省的另一份疑似Pr病料以及闽A（MinA）株细胞毒进行PCR扩增，均扩增到了全长目的基因片段，将三份扩增产物分别克隆于pMD 18-T 载体，对重组质粒作PCR、酶切分析和序列测定，证实了克隆片段的特异性。重组质粒分别命名为MinA-PK、YuA-PK和YuB-Pk。三个序列已被Genebank收录，收录号分别为：AY590265、AY644398、AY644399。
借助生物学分析软件，将 Prv MinA株,YuA株和 YuB株 PK基因的核苷酸序列与已被Genebank收录的 Prv Er 株、NIA-3 株及Ka株的 PK 基因核苷酸序列进行同源性和差异性比较，六个 Prv 毒株 PK 基因核苷酸序列间具有很高的同源性，最低者为YuA株和Ka株，其同源性为97.1％；YuA株在282位比其它毒株少一个G ，导致缺失突变，93位以后氨基酸序列的同源性大大降低，该毒株与MinA株，YuB株，Er株，Nia-3和Ka株同源性分别降为30.7％，30.7％，30.2％，29.5％,19.5％，明显低于其它组间的最低值 86.1％，分离毒对家兔的不致病性可能与该位点的缺
失突变有一定关系。以NiA-3株为标准，在其 238 至 249位是一个高变区，在 1081至 1098 位有一个高变区，这两个高变区没有打乱读码框，也没有破坏PK激酶的保守氨基酸位点，推测这两个区域对于PK激酶结构的稳定性和功能的发挥是非必需的。
本实验为了解河南省的Prv特性、分子流行病学、毒株之间的进化关系、进一步了解PK基因的功能和构建PrvPK缺失株奠定了基础，并为用Prv PK基因构建载体制备基因工程多价苗提供了新的思路和参考依据。
A strain of virus was isolated from a pig farm in Henan.The isolate was continually inoculated 4 generations onto PK15 cell and induced typical cytopathogenic effect(CPE).Three rabbits were injected with the isolates and no clinical sign was observed.The titers of the antibody to Prv in rabbits were 1:4、1:4 and 1:16 by LA 20 days post-challenge. The DNA extracted from the isolated virus was subjected to PCR amplifying ,the right fragment was amplified.All of the evidence indicated that the isolated virus was pseudorabies virus,and it was named strain YuA of pseudorabies virus.
A pair of primer were synthesized according the PK gene sequence of strains Er and NIA-3 published on GeneBank.PK genes of Prv strains MinA、YuA and YuB were amplified and cloned into vector pMD 18-T .These rocombinant plasmids were identified by restriction enzyme analysis and sequencing.
By means of molecular biology software,the PK genes of 3 Prv strains YU-A,Yu-B and Min-A were compared with that of another 3 strains(Er,NIA-3 and Ka) of Prv and were multialigmented.The results indicated that the identities between the 6 strain was high.The lowest identities (97.1% )was between strains YuA and Ka .A deletion of base G at the site 282th in Prv strain YuA accounted the evident difference in the amino acid sequence from the 93th Aa of YuA strain from the other strains.
The deduced amino acid identities of strain YuA with strains MinA,YuB,Er,NIA-3 and Ka were 30.7%,30.7%,30.2%,29.5%and19.5%,respectively, much less than the lowest identity (86.1% )between the other strains.The mutant of PK gene of strain YuA may have some relation with the rabbits’ showing no clinical sign.The nucleotide sequence analysis of the PK gene of the Prv strains revealed that there are 2 highly variable regions which lie on 238-249 nt and 1081-1098 nt in their nucleotide sequences.
利用上述引物，对来自河南省的另一份疑似Pr病料以及闽A（MinA）株细胞毒进行PCR扩增，均扩增到了全长目的基因片段，将三份扩增产物分别克隆于pMD 18-T 载体，对重组质粒作PCR、酶切分析和序列测定，证实了克隆片段的特异性。重组质粒分别命名为MinA-PK、YuA-PK和YuB-Pk。三个序列已被Genebank收录，收录号分别为：AY590265、AY644398、AY644399。
借助生物学分析软件，将 Prv MinA株,YuA株和 YuB株 PK基因的核苷酸序列与已被Genebank收录的 Prv Er 株、NIA-3 株及Ka株的 PK 基因核苷酸序列进行同源性和差异性比较，六个 Prv 毒株 PK 基因核苷酸序列间具有很高的同源性，最低者为YuA株和Ka株，其同源性为97.1％；YuA株在282位比其它毒株少一个G ，导致缺失突变，93位以后氨基酸序列的同源性大大降低，该毒株与MinA株，YuB株，Er株，Nia-3和Ka株同源性分别降为30.7％，30.7％，30.2％，29.5％,19.5％，明显低于其它组间的最低值 86.1％，分离毒对家兔的不致病性可能与该位点的缺
失突变有一定关系。以NiA-3株为标准，在其 238 至 249位是一个高变区，在 1081至 1098 位有一个高变区，这两个高变区没有打乱读码框，也没有破坏PK激酶的保守氨基酸位点，推测这两个区域对于PK激酶结构的稳定性和功能的发挥是非必需的。
本实验为了解河南省的Prv特性、分子流行病学、毒株之间的进化关系、进一步了解PK基因的功能和构建PrvPK缺失株奠定了基础，并为用Prv PK基因构建载体制备基因工程多价苗提供了新的思路和参考依据。
A strain of virus was isolated from a pig farm in Henan.The isolate was continually inoculated 4 generations onto PK15 cell and induced typical cytopathogenic effect(CPE).Three rabbits were injected with the isolates and no clinical sign was observed.The titers of the antibody to Prv in rabbits were 1:4、1:4 and 1:16 by LA 20 days post-challenge. The DNA extracted from the isolated virus was subjected to PCR amplifying ,the right fragment was amplified.All of the evidence indicated that the isolated virus was pseudorabies virus,and it was named strain YuA of pseudorabies virus.
A pair of primer were synthesized according the PK gene sequence of strains Er and NIA-3 published on GeneBank.PK genes of Prv strains MinA、YuA and YuB were amplified and cloned into vector pMD 18-T .These rocombinant plasmids were identified by restriction enzyme analysis and sequencing.
By means of molecular biology software,the PK genes of 3 Prv strains YU-A,Yu-B and Min-A were compared with that of another 3 strains(Er,NIA-3 and Ka) of Prv and were multialigmented.The results indicated that the identities between the 6 strain was high.The lowest identities (97.1% )was between strains YuA and Ka .A deletion of base G at the site 282th in Prv strain YuA accounted the evident difference in the amino acid sequence from the 93th Aa of YuA strain from the other strains.
The deduced amino acid identities of strain YuA with strains MinA,YuB,Er,NIA-3 and Ka were 30.7%,30.7%,30.2%,29.5%and19.5%,respectively, much less than the lowest identity (86.1% )between the other strains.The mutant of PK gene of strain YuA may have some relation with the rabbits’ showing no clinical sign.The nucleotide sequence analysis of the PK gene of the Prv strains revealed that there are 2 highly variable regions which lie on 238-249 nt and 1081-1098 nt in their nucleotide sequences.
引文
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[2] 苏鑫铭.伪狂犬病病毒上海株TK基因缺失株的构建及其生物学特性初步研究.南京农业大学.
[3] 范伟兴，刘文强摘译.伪狂犬病病毒的分子致病机理的研究进展[J].预防兽医学进展,2001，3（3）：45-48.
[4] 秦春圃.伪狂犬病毒的结构与功能[J].生物技术通报,2003，3：5.
[5] 吴清民.兽医传染病学[M].北京:中国农业大学出版所.2002.
[6] 姜炎，陈溥言.伪狂犬病病毒的囊膜糖蛋白gG的研究进展[J].中国兽医杂志，200238（12）：31～33.
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