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目的构建空肠弯曲菌cadF基因重组表达质粒pET30a(+)-cadF,分析其在大肠埃希菌中的表达情况及其融合蛋白的抗原性。方法用PCR方法扩增cadF基因,构建重组克隆质粒和表达质粒,经菌落PCR、双酶切和测序鉴定后在E.coli(DE3)原核表达系统中诱导表达,SDS-PAGE和飞行时间质谱鉴定表达蛋白,Western blot分析其抗原性。结果含重组表达载体pET-30a(+)-cadF中的cadF基因序列经测序证实与出发菌株cadF基因序列100%同源,并成功诱导表达CadF蛋白,Westernblot显示该CadF融合蛋白能被抗空肠弯曲菌多克隆兔血清识别。结论成功构建了空肠弯曲菌cadF基因重组表达载体,表达产物具有抗原性。
Objective To construct the recombinant expression plasmid pET30a (+) - cadF of Campylobacter jejuni and analyze its expression in Escherichia coli and the antigenicity of its fusion protein. Methods The cadF gene was amplified by PCR, and the recombinant plasmid was constructed. The recombinant plasmid was induced by E. coli (DE3) prokaryotic expression system after colony PCR, double enzyme digestion and sequencing. SDS-PAGE and time of flight mass spectrometry The protein was expressed and its antigenicity was analyzed by Western blot. Results The cadF gene sequence of the recombinant expression vector pET-30a (+) - cadF was confirmed to be 100% homologous to the cadF gene of the original strain by sequencing and successfully expressed CadF protein. Western blot showed that the CadF fusion protein could be expressed in anti - jejunal Bacterial polyclonal rabbit serum identification. Conclusion The recombinant expression vector of Campylobacter jejuni cadF gene was successfully constructed and the expressed product was antigenic.