禽多杀性巴氏杆菌P1059成熟黏附蛋白的原核表达、纯化和抗原性检测

Prokaryotic Expression and Purification of a Mature Adhesive Protein of Avian Pasteurella multocida P1059 and Detection of Its Antigenicity

目的:建立rCPM36在大肠杆菌中的表达体系,纯化表达产物并检测其抗原性。方法:运用PCR方法从禽多杀性巴氏杆菌国际标准株P1059基因组中扩增出编码36kDa成熟黏附蛋白的cpm36基因,构建原核表达载体pQE30-cpm36,转化到大肠杆菌M15中并诱导表达目的蛋白,用镍离子螯合层析柱纯化目的蛋白及制备其抗体,Western blot分析其抗原性。结果:SDS-PAGE结果显示目标蛋白以可溶性形式表达在大肠杆菌M15细胞质中,其相对分子质量为37kDa,Westernblot结果表明表达蛋白具有良好的抗原性。结论:成功构建出原核表达载体并实现了目的蛋白表达,用镍离子螯合层析柱纯化得到具有抗原性的蛋白,为进一步开展禽多杀性巴氏杆菌黏附因子和保护性抗原的研究奠定基础。

Objective： To express and purify recombinant mature adhesive protein （rCPM36）of avian Pasteurella multocida type strain P1059 and detect its antigenicity. Methods： The cpm36 gene, encoding a mature adhesive protein without signal peptide was amplified by PCR from genomic DNA of avian Pasteurella multocida strain P1059, PCR product was cloned into the expression vector pQE30 to constructed recombinant plasmid pQE30-cpm36 and then transformed into E. coli M15, inducing it with 0.2mmol/L IPTG for 4 hours. The expression and solubility of recombinant protein was detected by SDS-PAGE. The interest protein was purified by Ni-NTA affinity chromatography and its antigenicity was detected Western blot analysis. Results： SDS-PAGE showed that the 37kDa recombinant protein was successfully expressed in E. coli after IPTG inducing. In Western blot analysis, rabbit antiserum against rCPM36 reacted with rCPM36, it demonstrated that the recombinant protein is an antigenic protein. Conclusion： rCPM36 is successfully expressed in E. coli. The interest protein is successfully purified by Ni-NTA affinity chromatography and is detected its antigenicity. The rCPM36 protein might be a useful vaccine candidate antigen for avian Pasteurella multocida.