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目的构建与EPEC粘附相关蛋白IntiminC300、EspA、BfpA融合表达载体,并进行表达。方法用PCR方法从EPEC染色体中调取intiminC300、espA、bfpA基因,用基因重组的方法把3个基因片段用2个(Gly4Ser)3连接肽串联克隆到载体pQE30的多克隆区,转化宿主菌M15,用IPTG诱导表达,用SDS-PAGE和Western blot检测表达的融合蛋白。结果酶切和测序表明成功构建了融合表达载体,SDS-PAGE检测表达蛋白的分子质量单位分别为36、43和76kd,与理论值相符,Western blot显示表达的融合蛋白均能被抗His标签抗体识别。结论本研究成功构建了EPEC粘附相关融合蛋白表达载体,并表达目的蛋白,为进一步研究融合蛋白的免疫原性奠定了基础。
Objective To construct fusion expression vectors of IntiminC300, EspA and BfpA, which are related to the adhesion of EPEC. Methods The intiminC300, espA and bfpA genes were extracted from the chromosomes of EPEC by PCR. Three gene fragments were cloned into the polyclonal region of pQE30 vector by two recombinant vectors (Gly4Ser) 3 by gene recombination, and transformed into the host cell M15 , Induced with IPTG, and the expressed fusion protein was detected by SDS-PAGE and Western blot. Results The results of enzyme digestion and sequencing showed that the fusion expression vector was successfully constructed. The molecular mass units of the expressed protein detected by SDS-PAGE were 36, 43 and 76 kd, respectively. Consistent with the theoretical value, Western blot showed that the expressed fusion protein could be detected by anti-His- Recognize. Conclusion This study successfully constructed EPEC adhesion-related fusion protein expression vector and expressed the target protein, which laid the foundation for further study on the immunogenicity of the fusion protein.