【摘要】 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。更多还原

【Abstract】 Objectives To construct a vector expressing the fusion protein of IntiminC300-EspA-BfpA,which is related to enteropathogenic Escherichia coli(EPEC) adhesion,and to express that fusion protein.Methods The genes to express IntiminC300,EspA and BfpA,were amplified from the genome of EPEC joined by a DNA linker encoding(Gly4Ser)3 and cloned into the pQE30 multiple cloning site.The plasmids were then transformed into M15 and the fusion protein was detected with SDS-PAGE and Western blotting.Results The vector was successfully constructed according to sequence and restriction enzyme digestion analysis.Induced by IPTG,the fusion proteins appeared as bands at 36,43,and 76 ku in SDS-PAGE,and special reaction bands to anti-His target antibody were observed in Western blotting.Conclusion The plasmid pQE30-IntiminC300-EspA-BfpA was successfully constructed and the fusion protein was expressed.This work has laid the foundation for further research on the immunogenicity of the fusion protein.更多还原