H9N2亚型禽流感病毒血凝素基因的克隆及原核表达

Clone and Procaryotic Expression of Hemagglutinin Gene of H9N2 Avian Influenza Virus

据GenBank收录的H9N2亚型禽流感病毒血凝素(HA)基因序列设计并合成引物,以H9N2亚型禽流感病毒RNA为模板,用RT-PCR方法扩增了预计约1 700 bp的HA基因,将此扩增产物克隆进pMD18-T载体,采用限制性酶切及序列测定鉴定阳性重组克隆子。结果表明HA基因长为1 683 bp。基于HA信号肽在表达中的负作用,研究通过基因工程手段缺失HA蛋白位于起始的信号肽的编码序列,获得了缺失HA蛋白信号肽的HA基因,将其亚克隆到pGEX-KG中,与GST融合表达。SDS-PAGE显示:融合表达的蛋白分子量约为90 kDa。

According to HA gene sequence of H9N2 avian influenza virus collected by GenBank, We designed and synthesized primers, 1700bp fragment was amplified from H9N2 AIV RNA by RT-PCR and cloned into the pMD18-T vector. Restrictive enzyme and sequence were used to determinate positive recombinant cloning. The results showed that HA gene consisted of 1683bp. Based on the negative function of signal peptide of HA gene in expression, we lacked the signal peptide of HA gene which lies in initial coding sequence through the genetic engineering means and obtained the HA gene which lacked signal peptide, then partial fragment of HA was subcloned into prokaryotic expression vector pGEX-KG, and was fusion expressed with GST. SDS-PAGE showed that protein molecular weight was about 90kDa.