猪繁殖与呼吸综合征病毒ORF5基因在昆虫细胞中的高效融合表达

Fusion Expression of the ORF5 Gene of Porcine Reproductive and Respiratory Syndrome Virus in Insect Cells

对杆状病毒Bac to Bac表达系统的转座质粒pFastbac1进行改造 ,即在其多角体蛋白启动子下游插入谷胱苷肽 S 转移酶 (glutathione S transferase ,GST)基因 ,构建GST融合表达转座质粒pFGST。通过转座和转染Sf9细胞 ,证实该系统能高水平表达GST。采用PCR方法从pMT gp5 1质粒中扩增截去N端信号肽序列的猪繁殖与呼吸综合征病毒 (PRRSV)YA株ORF5基因 ,并将截短的ORF5基因片段克隆到pFGST中 ,使之与GST融合 ,构建的重组转座质粒pFGST 5 3转染DH1 0Bac ,提取大分子BacmidDNA ,转染Sf9细胞 ,获得能表达融合蛋白的高滴度重组病毒rvGST5 3。rvGST5 3感染Sf9细胞 ,SDS PAGE和Western印迹分析表明 :与GST融合的ORF5基因在Sf9细胞中获得高效表达 ,表达产物分子量为 45kD ,能与抗PRRSVE蛋白单克隆抗体发生特异性反应。将表达产物免疫小白鼠 ,经间接免疫荧光检测 ,免疫血清能使PRRSVYA株感染的MARC 1 45细胞呈较强的荧光着色 ,证实表达的融合蛋白具有良好的免疫原性。

A 0.75kb fragment containing the complete cDNA of glutathione-S-transferase(GST) and a modified thrombin cleavage sites were amplified and cloned into pFastBac1, the donor plasmid of Bac-to-Bac baculovirus expression systems, downstream from the polyhedrin promoter(pPolh), resulting in the GST fusion transposition plasmid pFGST. After transposition and transfection, SDS-PAGE analysis showed that the developed GST fusion expression system can highly express GST. The signal sequences of the major structural protein gene ORF5 of porcine reproductive and respiratory syndrome virus (PRRSV) strain YA was removed by PCR and the truncated ORF5 gene was inserted into pFGST and fused with GST to generate the transposition plasmid expressing GST-ORF5 fusion protein. The recombinant baculovirus rvBacGST53 was obtained by transposition and transfection. GST-ORF5 fusion protein was identified by SDS-PAGE and Western blot. The fusion protein is 45kD and is specific to the monoclonal antibody against the E protein of PRRSV. The fusion protein was directly injected into mice and the anti-sera can react with the PRRSV-infected MARC-145 cells by indirect immunofluorescent assay(IFA), indicating that the fusion protein have better immunogenicity.