西瓜花叶病毒外壳蛋白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)陕西分离物外壳蛋白(CP)基因,大小为843 bp.将CP基因克隆到pMD18-T Simple Vector,测序分析发现与各个国家CP核苷酸和氨基酸同源性分别为93.0%~95.0%和96.5%~98.6%.将CP基因定向插入EcoR I/SalI切开的pET30a中,构建了原核表达载体pET30-WCP,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37 kD的CP蛋白.通过不同时间诱导发现,加入IPTG 4 h后蛋白开始表达,8 h后表达量比较大.以诱导的蛋白为抗原免疫家兔,制备了WMV CP抗血清,ELISA法测其效价为1/6 400,Western blot分析能与CP发生血清学反应.

Cloning and Prokaryotic Expression of Coat Protein Gene from Watermelon Mosaic Virus

Coat protein(CP) gene from watermelon mosaic virus Shaanxi isolate was obtained by RT-PCR,it has 843 nts.CP gene has been cloned into pMD18-T Simple Vector,and sequenced.Sequence analysis indicates that the nucleotide and amino acid homologies are 93.0%~95.0% and 96.5%~98.6% aligned with WMV CP gene of other countries,respectively.CP gene is inserted into pET30a digested by EcoR I/Sal I,the expression vector of pET30-WCP is constructed,and transformed into E.coli BL21.Induced by IPTG for 2~8 h,the CP protein with molecular weight of about 37 kD is successively expressed.Comparison of different induce time finds that the CP protein begins to express for 4h after induced by IPTG,and the yields are the largest for 8 h.The purified protein induced as antigen is used to immunize the rabbit,and the antiserum of CP is prepared whose titer measured by ELISA is 1:6 400;western blot analysis indicates that the antiserum could serologically react on CP.