产气荚膜梭菌ε毒素突变基因的表达及其抗体中和效价测定

Expression of mutant ε-toxin gene of Clostridium perfringens and determination of neutralization antibody titer to ε-toxin

为研究突变产气荚膜梭菌ε毒素(mε)的抗体是否具有中和作用,本研究以毒素D型产气荚膜梭菌全基因组D N A为模板,应用重叠延伸PCR技术(SO E-PCR)将ε毒素第106位的组氨酸残基定点突变为脯氨酸残基,扩增出mε基因,使其丧失生物学毒性而保持免疫原性。以pET-28a为表达载体,构建重组表达质粒pET-mε,并转化大肠杆菌,经IPTG诱导表达重组mε蛋白。SDS-PAGE及w estern blot分析显示,重组蛋白约39 ku,主要以包涵体形式存在。将重组蛋白免疫家兔,所制备的产气荚膜梭菌mε毒素蛋白的抗血清经10倍稀释后,与一个绝对致死量的产气荚膜梭菌培养上清等体积混合时,能够有效中和ε毒素。这些结果表明,重组突变的ε毒素具有较强的抗原性,为产气荚膜梭菌候选亚单位疫苗的研究及其血清学检测方法的建立奠定了基础。

To investigate the neutralization of e-toxin with the antibody induced with the mutant e-toxin of Clostrdium perfringens, the mutant ε-toxin （me） gene was amplified from C.perfingens type D genomic DNA with the mutant primers by SOE-PCR, in which the mutant of an amino acid was created （HislPro） to inactivate toxicity of the protein. The mε gene was cloned into pET-28a vector and expressed in E.coli. The SDS-PAGE and western blot analysis showed that the recombinant me protein was about 39 ku and mainly in the form of inclusion body. The neutralization test results demonstrated that the LD100 of ε-toxin was able to be completely neutralized by the 10 times diluted antiserum prepared using the purified recombinant mutant ε-toxin in rabbit. Those results indicated that the recombinant mutant ε-toxin possessed strong antigenicity which could be used as candidate subunit vaccine and the antigen for development of serologic diagnosis method to C.perfringens infection.