棉铃虫单核衣壳核型多角体病毒HaSNPV orf101的原核表达及抗体制备  

Prokaryotic Expression of orf101 from HaSNPV and the Antibody Preparation

作　　者：李志远[1] 邱小慧[1] 周玥[1] 钱丽莹[1] 徐旭士[1] 

机构地区：[1]南京师范大学生命科学学院,江苏南京210046

出　　处：《安徽农业科学》2008年第35期15391-15393,15396,共4页Journal of Anhui Agricultural Sciences

基　　金：教育部基金项目(2006104SBJ0123)

摘　　要：[目的]摸索棉铃虫单核衣壳核型多角体病毒(HaSNPV)orf101编码蛋白原核表达条件并制备其多克隆抗体。[方法]根据HaS-NPVorf101序列,设计引物,引入适当的酶切位点,利用PCR扩增出基因片段并将其克隆至pGEM-TEasy载体,然后亚克隆至原核表达载体pGEX-KG,检测在不同条件下融合蛋白的表达产量及其折叠性质。原核表达产物经SDS-PAGE分离纯化,免疫新西兰大白兔制备多克隆抗体。[结果]在IPTG诱导的条件下,融合蛋白GST-HA101可在大肠杆菌BL21中以包涵体形式高效表达,表达产物的大小为55.8 kDa。制备的多克隆抗体经1∶8 000倍稀释后用于Western Blot分析,获得特异性显色信号。[结论]原核表达蛋白及其多克隆抗体的获得为深入研究Ha101的基因功能打下了基础。[ Objective] This study aimed to seek the optimum expression conditions of HaSNPV orf101 coding protein in prokaryotic cells, and to prepare the anti-HA101 multiclonal antibodies. [ Method] The orf101 was amplified by PCR from the genome DNA of HaSNPV. The PCR products was cloned into pGEM-T Easy vector, then it was subeloned into the GST-fused expression vector pGEX-KG. The expressed yield and folded property of the GST fusion protein were detected in different conditions. The protein was purified by SDS-PAGE. New Zealand white rabbit was immunized with the purified protein. [ Result] After IPTG induction, the E. coil BL21 containing recombinant plasmids expressed a considerable fusion protein of GST-HA101 in the form of inclusion body. The molecular weight of the fusion protein was 55.8 kDa, which was in agreement with the anticipation. Western blot analysis using the muhiclonal antibodies with 1：8 000 diluted showed a specific reaction to GST-HA101 fusion protein expressed in E. coil BL21. [ Conclusion] The prokaryotie expression protein and its antibodies made it possible to analyze the function of HA101 in infected insect cells in the future.

关 键 词：棉铃虫单核衣壳核型多角体病毒 orf101 克隆 表达 抗体 

分 类 号：S188[农业科学—农业基础科学]