A组轮状病毒SA11VP6基因的克隆和表达

CLONING AND EXPRESSION OF THE MAJOR CAPSID PROTEIN VP6 OF SIMIAN ROTAVIRUS STRAIN SA11

从ＳＡ１１ＶＰ６基因全序列克隆开始，设计一对两端带有酶切位点的引物，逆转录ＰＣＲ扩增出ＶＰ６全基因ｃＤＮＡ。经酶切后插入ｐＵＣ１９，构建了ＶＰ６全基因克隆ｐＲＡ６。再经酶切后插入痘苗病毒载体质粒ｐＪＳＡ１１７５中。利用Ｌｉｐｏｆｅｃｔｉｎ寻人ＴＬ－１４３细胞，利用ＴＫ基因和Ｌａｃ基因作为重组病毒的筛选标记。表达产物用单克隆抗体ＥＬＩＳＡ法检测，发现细胞培养上清和细胞裂解液都是阳性。Ｗｅｓｔｅｒｎｂｌｏｔ显示，表达产物分子量大小与天然ＶＰ６一致。

otaviruses are the major pathogens that cause life threatening dirarrhea in young childrenand animals .The gene encoding the major inner capsid protein VP6 of the simian rotavirusstrain SA11 was cloned and inserted into the thymidine kinase (TK)gene of vaccinia virus un-der the control of the 7.5 kD promoter. The VP6 was expressed successfully when 143 TK-cells were infected with the recombinant virus containing SA11 gene 6 insert,Reactivity with monoclonal antibody suggests that VP6 expressed intracellularly or found in media, maintainedits native antigenic determinants.