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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 the genetically engineered bacteria of Clostridium perfringens alpha toxin (CPA) and Escherichia coli heat labile enterotoxin subunit (LTB) fusion protein with good immunogenicity. Methods The recombinant plasmid pCPA-LTB was transformed into Escherichia coli BL21 (DE3) and induced by IPTG. The expressed protein CPA-LTB was detected by SDS-PAGE and Western blot. Mice were immunized orally or intraperitoneally with the expression product and challenged with tail vein after 21 days to observe the immunogenicity and immunoprotection of the fusion protein. Results The fusion gene CPA-LTB was expressed in Escherichia coli BL21 (DE3). The molecular mass unit of expressed product was 66ku. The expressed fusion protein can be recognized by anti-LT serum and type A C. perfringens antitoxin sera with CPA and LTB antigenicity. The LTB portion of the fusion protein enhances the immunogenicity of CPA and acts as an adjuvant for immunity. Conclusion The fusion protein CPA-LTB can be expressed in Escherichia coli BL21 (DE3), and the expressed CPA-LTB fusion protein has good immunogenicity.