Nazierbieke Wulumuhan, Yan Fang, He Cui, Zhang Lei, Borrathybay Entomack
Laboratory of Bioengineering, College of Biology and Environmental Sciences, Jishou University, Jishou 41600, China.
Sheng Wu Gong Cheng Xue Bao. 2008 Aug;24(8):1446-53.
The cp36 gene encoding an adhesive protein was amplified by PCR from genomic DNA of rabbit P. multocida C51-3 strain, and cloned into the pMD18-T vector and then sequenced. The mature adhesive protein without a signal peptide of cpm36 gene was amplified by PCR from the recombinant plasmid pMD18-cp36, then cloned into the prokaryotic expression vector pQE30 to provide a recombinant plasmid pQE30-cpm36. The recombinant protein of CPM36 was produced in Escherichia coli M15 harboring the recombinant plasmid pQE30-cpm36 by IPTG induction, and the recombinant protein purified by the affinity chromatography with Ni(2+)-NTA resin. The sequence analyses showed that the ORF of cp36 gene was 1032 bp in length, and DNA homology of the cp36 genes between the C51-3 strain and the previously reported different serotype strains of P. multocida in GenBank was 76.9 to 100%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 37 kD, and the Western blotting analysis demonstrated that the recombinant protein CPM36 and native 36 kD protein of C51-3 were recognized specifically by an antiserum against the recombinant protein, suggesting that the recombinant protein is an antigenic protein.
通过PCR从兔多杀性巴氏杆菌C51 - 3株基因组DNA中扩增出编码黏附蛋白的cp36基因，将其克隆到pMD18 - T载体中并测序。从重组质粒pMD18 - cp36中通过PCR扩增出不含信号肽的cpm36基因成熟黏附蛋白，然后克隆到原核表达载体pQE30中，构建重组质粒pQE30 - cpm36。携带重组质粒pQE30 - cpm36的大肠杆菌M15经IPTG诱导表达CPM36重组蛋白，并用Ni(2 +)-NTA树脂亲和层析法纯化重组蛋白。序列分析表明，cp36基因的开放阅读框长度为1032 bp，C51 - 3株与GenBank中先前报道的多杀性巴氏杆菌不同血清型菌株的cp36基因DNA同源性为76.9%至100%。SDS - PAGE分析显示有一条分子量为37 kD的单一融合蛋白条带，Western blotting分析表明重组蛋白CPM36和C51 - 3株天然36 kD蛋白能被抗重组蛋白抗血清特异性识别，提示该重组蛋白是一种抗原蛋白。
