鸭疫里默氏杆菌OmpA基因的克隆与表达

Cloning and expression of the ompA gene of Riemerella anatipestifer

利用本实验室分离、鉴定并保存的鸭疫里默氏杆菌血清1型湖北云梦分离株(RA-YM),根据GenBank上的序列设计了1对引物,用PCR方法扩增了RA-YM株的主要外膜蛋白OmpA基因,并克隆到pMD18-T载体,序列测定结果表明OmpA基因全长1152bp,序列同源性分析表明该基因相当保守,与其他不同RA菌株OmpA基因之间的核苷酸序列同源性为93%～97%。用pGEX-KG构建了原核表达载体pKG-OmpA,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30%,主要为包涵体形式。SDS-PAGE电泳结果显示表达的融合蛋白大小约为68000,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 OmpA gene of Riemerella anatipestifer（RA）,the OmpA gene was amplified from the isolated YM strain of RA （RA-YM strain） by PCR. The PCR amplified DNA fragment was cloned into pMD18-T. The sequencing results indicated that this gene was 1 152 bp and quite conservative. The result of sequence analysis of OmpA gene of RA-YM indicated that the homology among other RA strains was 93%-97%. The OmpA gene was ligated into pGEX-KG to get the expressing vector of pKG-OmpA which was then transformed into E. coli8 BL21. The result of SDS-PAGE showed the expressed protein was about 68 000,and the recombinant protein occupied 30% of total cell protein and in inclusion body form. Western-blot results indicated that this protein possesses biological activity.