猪传染性胃肠炎华毒弱毒株纤突蛋白B、C抗原位点片段基因的克隆与序列分析

Cloning and Sequencing of B、C Antigenic Sites Fragment of S Gene from Transmissible Gastroenteritis Attenuated Virus Strain H

猪传染性胃肠炎华毒 (TGEVH)是我国分离的代表毒株 ,H弱毒株是我国自行培育成功的具有良好免疫原性的疫苗株。应用RT_PCR方法扩增出 1.3kb的目的片段 ,包括S基因的编码的B、C两个主要抗原位点的片段 ,目的片段与pUC19质粒载体进行连接 ,转化 ,鉴定后得到阳性重组质粒 ,命名为pTS1,将重组质粒插入的目的基因片段进行序列分析和比较 ,其结果表明TGEVH株S基因 5′端B、C位点片段 1190bp的核苷酸序列与国外的TGEV毒株Miller、Purdue_115、FS772 / 70及台湾省TFI毒株序列均具有很高的同源性 ,达 94.72 %以上。推导的氨基酸序列同源性为 94.70 %以上。编码抗原位点C的序列与其它毒株完全一致 ,但在B位点发生改变 ,即第 97个氨基酸发生改变 ,由Trp突变为Ser。该核苷酸该片段全长 12 6 1bp ,包括起始密码子ATG及基因间的共同序列ACTAAAC。本研究首次报道了国内的TGEV分离株_H弱毒株S基因的RT_PCR扩增及其编码的B、C抗原位点片段的分子克隆及序列分析 ,为进一步揭示TGEV强弱毒差别的分子机制。

Chinese isolated Transmissible gastroenteritis virus(TGEV) attenuated strain H has good immunogenic.Antigenic sites B and C segments about 1.3kb of S gene was amplified from attenuated TGEV strain H by RT_PCR,The PCR products were purified and then cloned into plasmid pUC19,the recombinant plasmids were designated pTS1 and analyzed by endonecleoase digestion for proper inserts.The sequence analysis of the insert fragments in the recombinant pTS1 indicated that the TGE strain H shared more than 94.72% with miller、Purdue_115、FS772/70 and TFI. The amino acid sequence homology in this domain was above 94.70%.The amino acid residues that contribute to antigenic sites C didn't change,but the 97th amino acid residues Trp was substiuted by Ser in antigentic site B.We first report RT_PCR amplification antigenic sites B.C segments of S gene from Chinese isolated TGEV attenuated strain H,and the major antigenic sites fragments were cloned and sequenced.It provides a basis of the studies for the new type vaccine and molecular diagnosis.