猪流感病毒新型核糖体移码蛋白PA-X特有序列的原核表达、纯化及鉴定

PROKARYOTIC EXPRESSION, PURIFICATION AND IDENTIFICATION OF PA-X-SPECIFIC SEQUENCE OF SWINE INFLUENZA VIRUS

核糖体移码蛋白PA-X是流感病毒的一种新型蛋白,为了研究该蛋白对于猪流感病毒的生物学特性的影响,我们进行了PA-X特有序列的原核表达、蛋白纯化及其鉴定。通过RT-PCR和重叠PCR方法扩增PA-X特有序列的基因,利用基因重组技术构建原核表达载体,并进行菌液PCR和菌液测序鉴定,鉴定正确后克隆转化到BL21(DE3)中,进行蛋白表达及可溶性分析,同时用镍柱亲和层析法对其进行纯化。通过菌液PCR序列鉴定,PA-X特有序列的原核表达载体序列正确,编码框正确。转化BL21后目的蛋白表达成功,且大多数为可溶性蛋白。Western blot结果表明制备的多克隆抗体具有良好的免疫反应性。

The novel infl uenza A virus protein PA-X is a ribosomal frame-shifting product that modulates the host response and viral virulence. In the present study, PA-X protein was prokaryotically expressed, purified and characterized in order to investigate its biological functions. The PA-X coding gene was amplifi ed in RT-PCR and overlapping PCR. The prokaryotic expression vectors were constructed with the gene recombination technique and verifi ed using bacterium solution PCR and sequencing. The positive clones were transformed into E.coli BL21（DE3） and the expressed protein was analyzed for its solubility. The PA-X products were purifi ed using nickel column chromatography. The sequences of the plasmids and open reading frames were demonstrated to be completely correct. After transformation, these plasmids expressed the expected proteins with soluble nature. Subsequently, the PA-X protein was purifi ed and used in Western blot to react with the mouse antiserum against the H1N1 swine infl uenza virus. The positive reaction between the PA-X protein and mouse antiserum was observed.