副猪嗜血杆菌OMP P2基因的克隆与表达

Cloning and expression of the OMP P2 gene of Haemophilus Parasuis

利用已分离的菌株HB070412,根据NCBI上GenBank中的HPS序列(ZP_02477753)设计了1对引物,用PCR方法扩增了副猪嗜血杆菌外膜蛋白中的穿孔素前体蛋白P2基因(OMP P2),并克隆到载体pMD18-T(T-Vec-tor),序列测定结果表明OMP P2基因全长1 077bp,序列同源性分析表明该基因相当保守,与所报道的HPS OMPP2基因之间的核苷酸序列同源性为98.1%,氨基酸序列同源性为95.5%。用pGEX-6p-1构建了原核表达载体pGEX-P2,在大肠杆菌中进行了高效表达,表达蛋白约占菌体总蛋白的50%,主要为包涵体形式。SDS-PAGE电泳结果显示表达的融合蛋白约为65 000,Western blot结果表明该表达蛋白具有抗原性。

A pair of specific primers was designed based on the published sequences of the OMP P2 gene of Haemophilus Parasuis（HPS）,the OMP P2 gene was amplified from the isolated strain HB070412 of HPS by PCR.The PCR amplified DNA fragment was cloned into pMD18-T.The sequencing results indicated that this gene was 1 077 bp and quite conservative.The result of sequence analysis of OMP P2 gene of HB070412 indicated that the homology among other HPS strains would be from 95.5% to 98.1%.The OMP P2 gene was ligated into pGEX-6p-1 to get the expressing vector of pGEX-P2 which was then transformed into E.coli BL21.The result of SDS-PAGE shows the expressed protein is about 65 000,and the recombinant protein accounts for 50% of total cell protein and in inclusion body form.Western blot results indicate that this protein possesses biological activity.