西瓜花叶病毒HC-Pro基因在毕赤酵母中的分泌表达与功能研究

Research on Secretion Expression in Pichia pastoris and Function of the HC-Pro Gene of Watermelon Mosaic Virus

利用RT-PCR方法扩增出西瓜花叶病毒HC-Pro的基因,长度为1371bp,并构建了真核表达质粒pPIC9K-WHC。将重组质粒经SalⅠ单酶切后电转化Pichia pastoris GS115菌株,经PCR鉴定与G418、MD和MM培养基筛选,获得Mut+/His+表型高拷贝转化子。经1%甲醇诱导5d后,SDS-PAGE检测发酵液上清,在66kD处有一特异蛋白条带表达。Western blot鉴定表明,表达蛋白可以和HC-Pro蛋白抗血清发生结合反应。Far-Western blot证明该蛋白能与西瓜花叶病毒CP蛋白结合,支持了HC-Pro蛋白协助传毒的"桥梁"学说。

HC-pro gene of Watermelon Mosaic virus was obtained by RT-PCR was 1371bp in length. It was cloned into pPIC9K, then the eucaryotic recombinant expression plasmid pPIC9K-WHC was constructed. After being linearized with restriction endonuclease Sal I , the recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation. The high copy transformants with Mut^＋/His^＋ phenotype were selected by RT-PCR and screening on G418, MD and MM medium. Induced by methanol for 5 days, the culture supernatant was analyzed by SDS-PAGE, the results showed that a specific protein with a molecular weight of about 66kD was expressed. Western blot analysis proved that the expression protein could specifically bind to HC-Pro polyclonal antibody. Far western blot analysis proved that the expression protein could bind to coat protein, given support to ＂bridge＂ hypothesis that HC-Pro help aphid transmission of non-persistent viruses.