猪传染性胃肠炎病毒S蛋白主要抗原位点在昆虫细胞中的表达

Expression of the main sites of transmissible gastroenteritis virus spike protein in insect cells

用RT-PCR方法扩增出猪传染性胃肠炎病毒S基因主要抗原位点A、D,将其亚克隆至供体质粒pFastBac1,得到重组转移质粒pFastBac1-TS1,转化到Escherichia coliDH5α中,提取阳性克隆质粒再转化到含穿梭载体bacmid的E.coliDH10Bac中,发生转座作用,经蓝白斑筛选得到重组穿梭载体Bacmid-TS1,经PCR鉴定后再转染Sf9昆虫细胞,经空斑筛选得到纯化的重组病毒rAcV-Bac-TS1,经IFA、SDS-PAGE和Western-blot鉴定,重组杆状病毒TS1蛋白(相对分子质量约43 000)在Sf9昆虫细胞中得到表达。

The main sites of transmissible gastroenteritis virus spike gene A and D was amplified by RT-PCR and was cloned into the eontribution vector pFastBacl to get the recombinant plasmid pFastBacl-TS1, pFastBacl-TS1 was then introduced into Esche- richi, coil DHIOBac, which included a shuttle vector, bacmid. By site-specifie transposition, S gene sites A and D were integrated into baemid, and a recombinant shuttle vector was constructed, named Bacmid-TS1. The recombinant bacmid, which was identified by PCR, was transfected into St9 insect cells. And the pure recombinant baculovirus was obtained by plaque. Expression of TS1 protein （relative molecular mass about 43 000） was examined and identified by IFA, SDS-PAGE and Western-blot.