猪细小病毒NS_1基因的克隆及表达

Cloning and Expression of PPV NS_1 Gene

根据 Bergeron等报道的猪细小病毒基因组序列设计引物 ,在下游引物中引入 Bgl 酶切位点以便于构建表达载体。利用 PCR技术扩增得到 NS1 全基因 ,将其克隆到 p MD18- T载体中进行序列测定并分析 ,进而构建重组转移载体p Fast- NS1 ,在 DH1 0 Bac大肠杆菌中与改造过的苜蓿夜蛾核型多角体病毒 (Ac NPV )基因组 (Bacmid)发生同源重组 ,获得重组穿梭载体 Bacmid- NS1 ,经脂质体介导转染 Sf9细胞后 ,得到重组杆状病毒 (Ac NPV- NS1 )。SDS- PAGE、Western-blotting分析可见大小约为 84 0 0 0的特异性带 ,表明 NS1 蛋白在杆状病毒 Bac- To- Bac表达系统中成功地实现了表达 ,从而为研究其结构与功能创造了条件。

A pair of primers was designed according to the PPV genome sequence reported by Bergeron,and a BglⅡ site was introduced for development of expressing vector.The whole NS 1 gene was obtained by PCR,and cloned into pMD18 T to sequence.The recombinant transfer vector(pFast NS 1) was developed.Bacmid NS 1 was obtained in DH 10 Bac E.coli,through homologous recombination between pFast NS 1 and bacmid that is genome modified of Autogragha californica nuclear polyhedrosis virus(AcNPV).After transfection,the recombinant virus(AcNPV NS 1) was developed.The specific approximate 84 000 band was obtained by analysis of SDS PAGE and Western blotting.The results indicated that PPV NS 1 protein had been expressed successfully in baculovirus Bac To Bac expression system.