兔多杀性巴氏杆菌C51-3株黏附蛋白的表达、纯化及其抗原性检测

Expression and Purification of an Adhesive Protein of Rabbit Pasteurella multocida C51-3 and Detection of Its Antigenicity

应用PCR从兔多杀性巴氏杆菌C51-3株基因组DNA中扩增出编码36kD黏附蛋白的cp36基因，将其克隆到pMD18-T载体并对插入片段进行测序。以重组质粒pMD18-cp36为模板，用PCR扩增得到编码信号肽除外的成熟黏附蛋白基因cpm36，并克隆到原核表达质粒pQE30中，得到重组质粒pQE30-cpm36，转化大肠杆菌M15，在IPTG诱导下表达融合蛋白CPM36，经Ni^2＋-NTA亲和层析纯化。DNA测序结果表明cp36基因片段大小为1032bp，与已报道的16个血清型多杀性巴氏杆菌cp36基因的核苷酸序列比较，同源性在76．9％～100％之间。SDS-PAGE结果显示，表达分子量约为37kD的带有6xHis标签的CPM36蛋白，与预期分子量相符。Western blotting结果表明，抗重组蛋白抗体分别能与CPM36蛋白和多杀性巴氏杆菌36kD蛋白发生特异性反应，证明原核表达蛋白具有抗原性，为进一步开展多杀性巴氏杆菌免疫保护性抗原的研究奠定了基础。

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 Ni2^＋-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.