兔多杀性巴氏杆菌外膜蛋白OMPH1基因的克隆及原核表达

Cloning and Prokaryotic Expression of Outer Membrane Protein gene OMPH1 of Pasteurella multocida isolated from rabbit

本试验旨在克隆和表达兔多杀性巴氏杆菌外膜蛋白OMPH1基因,并对其表达产物的反应原性进行研究。以兔多杀性巴氏杆菌C51-2-499株总DNA为模板,进行外膜蛋白基因OMPH1的PCR扩增,得到了1条大小为1 041 bp的基因片段,克隆至pMD-18T中。测序分析证明,它与国内外报道的多杀性巴氏杆菌OMPH1基因的核苷酸序列相似性达99%以上。将该基因克隆到原核表达载体pET-30a(+)中,经酶切和测序分析表明,重组表达载体pET-30a(+)-OMPH1构建成功。将此重组质粒转化至大肠杆菌BL21(DE3)中,用IPTG进行诱导。SDS-PAGE检测结果证实,该基因在大肠杆菌中以融合蛋白的形式得到表达,表达产物的分子质量约47KD,与预期的结果一致。Western-blot分析表明,OMPH1重组表达蛋白具有反应原性,可被兔多杀性巴士杆菌阳性血清识别,为兔多杀性巴氏杆菌基因重组疫苗及新型诊断试剂的研究奠定了基础。

This experiment was conducted to clone and express the OMPH1 gene.Total DNA was extracted from Pasteurella multocida isolated from rabbit and used as template,the 1041 bp OMPH1 outer membrane protein gene was amplified by PCR,and was cloned into pMD-18 T vector.The sequencing result showed that the homology of the gene sequence with the reported OMPH1 gene from Pasteurella multocida reached 99%.The OMPH1 gene was cloned into the expression vector Pet30a（＋）,the digestion identification and sequencing analysis showed that the recombinant expression vector pET-30a（＋）-OMPH1 was successfully constructed.The plasmid pET-30a（＋）-OMPH1 was transformed E.coli BL21（DE3）,and induced by isopropyl β-D-thiogalactoside（IPTG）.The SDSPAGE results confirmed that the gene was expressed in a form of inclusion body in E.coli.,the molecular weights of the expressed product were about 47 KD,which was consistent with the expected results.Westernblot analysis showed that the recombinant expres.sion protein can be recognized by the positive serum of rabbit source Pasteurella multocida.This study establishes the foundation for the research in recombinant vectored vaccine and new diagnostic reagents of Pasteurella multocida.