肠出血性大肠杆菌O157∶H7紧密黏附素免疫保护性片段的基因克隆与表达

Gene cloning and prokaryotic expression of intimin and intiminC300 from EHEC O157∶H7

目的 克隆表达肠出血性大肠杆菌 (EHEC)O1 5 7∶H7紧密黏附素 (intimin)免疫保护性片段 (intiminC30 0 )。方法 设计引物采用PCR法自EHECO1 5 7∶H7基因组扩增紧密黏附素及其免疫保护性片段的编码基因eae与eaeC30 0 ,T A克隆后构建原核表达质粒pET 2 8a(+) eae及pET 2 8a(+) eaeC30 0 ,经测序鉴定后转化E .coliBL2 1 (DE3) ,IPTG诱导表达 ,PAGE检测。结果 PCR法自EHECO1 5 7∶H7基因组扩增出了约 2 80 0bp和 90 0bp的目的片段 ;原核表达质粒pET 2 8a(+) eae及pET 2 8a(+) eaeC30 0经酶切及测序鉴定与预期序列一致。转化E .coliBL2 1 (DE3)后IPTG诱导目的蛋白表达率约 2 5 %和 30 %;PAGE初步测定目的蛋白的相对分子质量 (Mr)约 96× 1 0 3和 35× 1 0 3,破菌后电泳证实目的蛋白均以包涵体形式表达。结论 EHECO1 5 7∶H7紧密黏附素免疫保护性片段经基因克隆后获得了较高的表达量 ,为进一步的生物学性质及免疫学特性研究奠定了基础。

Objective To clone the gene of intimin and intiminC300 of EHEC O157∶H7(eae and eaeC300). Methods The gene of intimin and intiminC300 were amplified from EHEC O157∶H7 chromosome by PCR and then cloned into pMD18-T vector. Thereafter, the genes were cut from pMD18-T vector and cloned into prokaryotic expression plasmid pET-28a(+), and recombinant plasmids were transformed into E.coli BL21(DE3). The protein of intimin and intiminC300 were isolated and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot. Results The gene of intimin and intiminC300 were successfully cloned into pET-28a(+). The molecular weight of the expressed products were 96kD and 35kD, and the expression rate were about 25% and 30% respectively. Conclusion Intimin and intiminC300 of EHEC O157∶H7 were successfully expressed in prokaryotic expression system.