西瓜花叶病毒HC-Pro基因的克隆与原核表达

利用RT-PCR方法获得了西瓜花叶病毒(WMV)陕西分离物HC-Pro基因,大小为1 371 bp.将HC-Pro基因克隆到pMD18-T Simple Vector,测序分析发现与其它国家HC-Pro核苷酸同源性为90.7%~94.8%.将HC-Pro基因定向插入EcoR I/Sal I切开的pET30a中,构建了原核表达载体pET30-WHC,转化大肠杆菌BL21.经IPTG诱导2~8 h后,成功表达了分子量约为57 kD的HC-Pro蛋白.通过不同时间诱导发现,加入IPTG 2 h后蛋白开始表达,继续诱导到8h后表达量变化不大.以诱导的蛋白为抗原免疫家兔,制备了HC-Pro蛋白的抗血清,ELISA法测其效价为1/6 400,Western blot分析能与HC-Pro发生血清学反应.

Cloning and Prokaryotic Expression of HC-Pro Gene from Watermelon Mosaic Virus

HC-Pro gene from watermelon mosaic virus shaanxi isolate is obtained by RT-PCR,it has137 1 nts.HC-Pro gene is cloned into pMD18-T Simple Vector,and sequenced.Sequence analysis indicate that the nucleotide homologies are 90.7%~94.8% aligned with WMV HC-Pro gene of other countries,respectively.HC-Pro gene is inserted into pET30a digested by EcoR I/Sal I,the expression vector of pET30-WHC is constructed,and transformed into E.coli BL21.Induced by IPTG for 2~8 h,the HC-Pro protein with molecular weight of about 57 kD is successively expressed.Comparison of different induce time found that the HC-Pro protein begin to express after induced by IPTG for 2 h,and the yields have no variation for 8 h.The purified protein induced as antigen is used to immunize the rabbit,and the antiserum of HC-Pro is prepared whose titer measured by ELISA is 1∶6 400;western blot analysis indicated that the antiserum could serologically reacted with HC-Pro.