融合基因CPA-LTB的原核表达及其免疫原性研究

Prokaryotic expression and immunogenicity of the CPA-LTB fusion gene

目的获得具有良好免疫原性的产气荚膜梭菌α毒素(CPA)与大肠埃希菌不耐热肠毒素B亚单位(LTB)融合蛋白的基因工程菌。方法将重组质粒pCPA-LTB转化大肠埃希菌BL21(DE3),用IPTG诱导表达,采用SDS-PAGE和Western blot检测表达蛋白CPA-LTB。用表达产物经口服或腹腔免疫小鼠,21d后尾静脉攻毒,观察融合蛋白的免疫原性和免疫保护性。结果融合基因CPA-LTB可在大肠埃希菌BL21(DE3)中表达,表达产物的分子质量单位为66ku。表达的融合蛋白可被抗LT血清和A型产气荚膜梭菌抗毒素血清识别,具有CPA和LTB抗原性。融合蛋白中的LTB部分增强了CPA的免疫原性,起到了免疫佐剂的作用。结论融合蛋白CPA-LTB能在大肠埃希菌BL21(DE3)中表达,表达的CPA-LTB融合蛋白具有良好的免疫原性。

Objective To obtain a recombinant strain expressing CPA-LTB fusion protein that possesses good immunogenicity.Methods The recombinant plasmid pCPA-LTB containing the CPA-LTB fusion gene was transformed into Escherichia coli BL21（DE3）.The CPA-LTB fusion protein was expressed by induction with IPTG and analyzed using SDS-PAGE and Western blotting.A crude preparation was then injected in mice.After 21 days,animals were challenged with a lethal dose of Clostridium perfringens type A toxin.Results The CPA-LTB fusion protein was expressed in the recombinant strain BL21（DE3）（pCPA-LTB） and had a relative molecular mass of about 66 ku.The protein was recognized by Clostridium perfringens type A antitoxin and anti-LT serum according to Western blotting.More importantly,the LTB portion of CPA-LTB can serve as an adjuvant and enhance the immunogenicity of CPA.Conclusion The fusion protein CPA-LTB was expressed in the recombinant strain BL21（DE3）（pCPA-LTB） and possessed good immunogenicity.