【摘要】 目的将流感病毒RNA聚合酶PA6亚基片段进行亚克隆、表达,获得重组的亚克隆多肽,为进一步研究PA6亚基功能奠定基础。方法以PA亚基cDNA为模板,用PCR方法扩增出PA6片段,应用定向克隆策略将扩增后片段与高效表达载体pQE32重组,阳性克隆重组质粒经酶切及测序鉴定,IPTG诱导各亚克隆系高效表达,优化表达条件。结果PCR法扩增出402bp的片段,酶切及测序鉴定获得正确的重组质粒,在E.coliM15中表达得到相对分子量约为17KD的重组蛋白,获得重组多肽。结论成功地亚克隆及表达了流感病毒RNA聚合酶PA6亚基。更多还原

【Abstract】 Objective To subclone influenza virus RNA polymerase PA6 ,construct the recombinant expression plasmid ,express it and gain recombinant polypeptides for providing the basis of functional study on thepolymerase.Methods The PA6 fragment was amplified by PCR ,the fragment was cloned into expressionvector pQE32 by means of directional strategy. The positive transformant was screened by agarose gel electrophoresis after endonuclease digestion and sequencing. The fusion protein was expressed in E. coli M15 ,the expression condition was optimized.Results The fragment of PA6 with size 402bp was amplified by PCR. Thecorrect recombinant plasmid was characterized by endonuclease digestion and sequencing. The recombinant protein were expressed in E.coli M15 with relative molecular weight 17 KD. Conclusion The influenza virus RNApolymerase PA6 is subcloned and expressed in E. coli successfully.更多还原