OEPR法构建mETX-mCPA表达质粒及其重组蛋白的免疫效果评价

Construction of plasmid expressing mETX-mCPA by OEPR method and immune effect of recombinant protein

为了确定产气荚膜梭菌ε毒素(ETX)和α毒素(CPA)无毒突变体作为亚单位疫苗同时预防D型和A型产气荚膜梭菌病的免疫效果,试验用点突变结合OEPR法构建重组质粒pET32a-mETX-mCPA。将阳性重组质粒转入E.coli BL21(DE3)中,以预先添加乳糖的自诱导培养基表达目的蛋白。经SDS-PAGE分析,其表达的蛋白相对分子质量约为92 000,与预期值一致。Western blot检测结果表明,重组蛋白可被A型产气荚膜梭菌阳性血清所识别。致死性试验结果表明,重组蛋白不致死小鼠。以铝胶为佐剂将重组蛋白免疫家兔,二免血清0.1 mL可分别中和3个小鼠MLD的A型毒素和100个小鼠MLD的D型毒素。本研究用OEPR法成功构建了无毒突变体pET32a-mETX-mCPA质粒,获得的重组蛋白在家兔上显示出良好的免疫效果,为研发A、D型产气荚膜梭菌二价亚单位疫苗奠定理论基础。

In order to determine the immune effect of the recombinant protein mETX-mCPA as a subunit vaccine for preventing diseases caused by Clostridium perfringens type D and A,the recombinant expression plasmid pET32 a-mETX-mCPA was constructed by OEPR(overlap extension PCR and recombination) and site-directed mutagenesis.The positive recombinant plasmid was transformed into E.coli BL21(DE3),and the target protein was expressed in the auto-inducing medium including lactose.The expression product was identified by SDS-PAGE and found to be 92 000 as expected and confirmed by Western blot with Clostriudm perfringens type A antisera.Recombinant protein could not make mice die.Upon immunization of rabbit twice using the recombinant protein with aluminium hydroxide gel,the second rabbit immune sera neutralization titers against Clostridium perfringens type A toxin and type D toxin were 3 and 100 respectively.The non-toxic mutant pET32 a-mETX-mCPA was successfully constructed by OEPR,the obtained recombinant protein showed a good immune effect,providing basic data for subsequent development of subunit vaccine.