【摘要】 目的:为便于研究鼠疫菌基因及其产物的功能,利用重组工程技术,建立一种表位标记细菌染色体基因的方法,以分析基因的表达规律。方法:将标签序列合成于引物中,通过融合PCR法将标签序列与抗性筛选基因连接,克隆到pBluescript载体,获得模板载体pBSMH。设计与待标记基因末端及其下游同源的引物,从模板载体上扩增标签与抗性基因的融合模块,获得两端带同源区的PCR产物,电击转化鼠疫菌感受态细胞。在λRed重组系统的辅助下,标签和抗性基因重组到目的基因的下游,得到表位标签与目的基因C末端融合的重组子。利用针对表位标签的单克隆抗体进行Westernblot分析,验证C末端标记目的蛋白的表达。结果:成功构建了组氨酸标签与抗性基因融合的质粒,以该质粒作为模板,扩增标记标签盒,对鼠疫菌的rpoS基因进行了标记,利用针对组氨酸标签的抗体能够检测到rpoS基因的表达。结论:基于重组工程的基因标记技术可以为基因功能研究提供一个有价值的研究手段。更多还原

【Abstract】 Objective: To facilitate the functional analysis of chromosomal genes and their products, the recombineering technique to epitope tagging of chromosomal genes of Y. pestis was adapted. Methods: The epitope tag was generated by primer annealing and then fused with resistance gene by fusion PCR. The epitope-resistance cassette was inserted into pBluecript, resulted in the template plasmid, pBS-MH. The tagging cassette for rpoS was obtained by PCR amplification from pBS-MH with primers containing homology specific to the target gene. PCR products were transformed into recombination competent cells and recombinants were selected. PCR and DNA sequencing were used to confirm the correct tagging event. The expression of the tagged protein was detected with Western blot by using monoclonal antibody to the epitope. Results: The template plasmid containing fusion of epitope and resistance gene was successfully constructed. The sigma factor gene, rpoS, was tagged with a myc-his tag at the COOH terminus. Expression of the tagged rpoS was successfully detected indirectly by the antibody against His tag. Conclusion: The chromosomal gene tagging by recombineering technique represents a powerful tool in the functional study of bacterial genes and their products.更多还原