猪传染性胃肠炎病毒TH-98株S基因的克隆与同源性比较

用猪睾丸 (Swine testicle,ST)细胞繁殖并扩增猪传染性胃肠炎病毒国内分离株 TH- 98,根据 Gen Bank中已发表的TGEV S基因 c DNA序列 ,利用 Oligo4.1程序软件设计并合成两对引物 ,进行逆转录聚合酶链式反应 (RT- PCR) ,扩增出2 .3kb和 2 .1kb两个片段 ,分别命名为 Sa与 Sb,先后插入到 p UC18质粒载体上的 Eco RI和 Pst I多克隆位点上 ,构建了重组质粒 p UC- S。根据 TGEV S基因位点和 p UC18的物理图谱 ,用相应的限制性内切酶进行酶切及套式 PCR方法进行鉴定、分析 ,证明克隆的 p UC- S为 TGEV S基因。同时对 p U C- S进行序列测定分析 ,并与来自美国、英国和日本的五个毒株进行核苷酸及编码氨基酸同源性比较 ,证明了 S基因的高度保守性

Cloning and comparison of homology of S gene from transmissible gastroenteritis virus isolate TH-98

In this assay,TGEV native isolate TH-98 was propagated and harvested on swine testicle(ST) monolayer cell.Two paris of primers were designed to amplify S gene by RT-PCR according to the published sequence of TGEV'S gene cDNA with Oligo4.1.The products of PCR were named Sa and Sb,of 2.3kb and 2.1kb respectively.Sa was inserted in EcoRI and KpnI sites after Sb was cloned in KpnI and PstI multiple cloning sites of the same pUC18 plasmid.The recombinant pUC-S plasmid was identified and analyzed by corresponding physical map of pUC18 plasmid,which was identified as S gene of TGEV.Recombinant pUC-S was sequenced and analyzed in comparison with other homologous strains,which derived from US,British and Japan.Gene sequence comparison demonstrated that TH-98 shared 99%,97%,97%,94%,and 98%identities with Purdue-115(US),Miller(US),TO14(Japan),FS772(British)and 96-1933(British)respectively and their amino acid homology were 99%,97%,96%,93%and97% correspondingly.Therefore,the results affirmed that S gene of different isolates of TGEV was extremely conservative.