禽多杀性巴氏杆菌X-73株omp H基因的克隆和表达

Cloning and Expression of omp H Gene of Avian Pasteurella Multocida Strain X-73

以禽多杀性巴氏杆菌国际标准株X-73基因组DNA为模板,采用PCR扩增得到编码信号肽除外的成熟外膜蛋白H的ompH基因,将ompH基因克隆至pMAL-p2X表达载体中,构建重组表达质粒pMAL-p2X-ompH,转化大肠杆菌BL21,用SDS-PAGE电泳检测IPTG诱导后的表达蛋白.DNA测序结果表明ompH基因片段大小为1 002 bp,与已报道的ompH基因的核苷酸序列完全相同、而SDS-PAGE结果显示表达分子质量约为78 ku,与预期的融合蛋白MBP-OmpH分子质量相符,表明成功构建出原核表达质粒并实现了目的蛋白表达,为进一步开展禽多杀性巴氏杆菌保护抗原的研究奠定基础.

The ompH gene encoding mature outer membrane protein H （OmpH） without a signal peptide was isolated from genomic DNA of avian P. multocida type strain X-73 by PCR. The BamH I and Xho I digested PCR product was cloned into prokaryotic expression vector pMAL-p2X to provide a recombinant plasmid pMAL-p2X-ompH. The fusion protein of MBP-OmpH was expressed in E. coli BL21 harboring the recombinant plasmid pMAL-p2X-ompH by IPTG induction,and the recombinant protein was detec- ted with SDS-PAGE. The sequence analysis showed that the ornpH gene coding the mature protein was 1 002 bp in length,and completely identical to the previously reported ompH gene of strain X-73.while the SDS-PAGE showed that a single protein band with molecular weight of 78 kDa in E. coli after IPTG inducing. The recombinant protein OmpH can be used as positive control on development subunit vaccine for fowl cholera.