禽流感病毒H5亚型HA1基因的原核表达及纯化

Expression and purification of HA1 gene H5 subtype from avian influenza virus

根据已报道的禽流感病毒H5亚型HA1基因设计1对引物,利用RT-PCR技术扩增目的基因片段,将扩增片段插入到pPROEX-HTb表达载体上,并对重组表达质粒进行PCR酶切鉴定,通过序列测定验证读码框,并在大肠埃希氏菌BL21(DE3)中进行表达.结果表明:扩增出的1 000bp的基因片段在大肠埃希氏菌中成功表达,蛋白产物约为38ku,经Western-blotting分析表明该融合蛋白具有很好的免疫原性.

According to published HA1 gene H5 subtype sequence of avian influenza virus（AIV） in Genbank,a pair of primers were designed and synthesized.Using the primers,a gene of approximately 1 000 bp in length was amplified by RT-PCR,then it was inserted into the plasmid vector pPROEX-HTb,the recombinant plasmid was identified by PCR and expressed in Escherichia coli.The results showed that an expected fusion protein of 38 ku in size was successfully expressed which had right immunity by western-blotting.