西瓜花叶病毒HC-Pro基因的克隆与原核表达  

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

作　　者：张建新[1] 刘起丽[2] 吴云锋[3] 

机构地区：[1]河南师范大学生命科学学院,河南新乡453007 [2]河南科技学院植物保护系,河南新乡453003 [3]西北农林科技大学植物保护学院,陕西杨凌712100

出　　处：《河南师范大学学报（自然科学版）》2009年第4期101-104,共4页Journal of Henan Normal University(Natural Science Edition)

基　　金：教育部长江学者和创新团队发展计划(200558);河南师范大学青年科学基金(2008qk16)

摘　　要：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1371bp．将HC—Pro基因克隆到pMD18-T SimpleVector，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EcoRⅠ／SalⅠ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2～8h后，成功表达了分子量约为57kD的HC-Pro蛋白．通过不同时间诱导发现，加入IPTG2h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1／6400，Westernblot分析能与HC-Pro发生血清学反应．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.

关 键 词：西瓜花叶病毒 HC-PRO 原核表达 抗血清 

分 类 号：S432.4[农业科学—植物病理学] Q93[农业科学—农业昆虫与害虫防治]