新型鸭细小病毒VP3基因的原核表达及多克隆抗体的制备  

Prokaryotic expression of VP3 gene of novel duck parvovirus and preparation of its polyclonal antibody

作　　者：李琦[1,2] 李传峰[2] 唐井玉 刘柱[2,4] 宫晓华[2,3] 陈宗艳[2] 王桂军[3] 吴润[1] 刘光清[2] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院上海兽医研究所,上海200241 [3]安徽农业大学动物科技学院,安徽合肥230036 [4]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《中国兽医科学》2016年第6期717-722,共6页Chinese Veterinary Science

基　　金：上海市科委科技创新医药与农业领域项目(13391901602);国家自然科学基金(31502068;No.31270194);农业部公益性行业科研专项(201303046);上海市闵行区高层次人才队伍建设专项资金

摘　　要：应用PCR方法扩增了新型鸭细小病毒(novel duck parvovirus,NDPV)的VP3基因,然后将其克隆到原核表达载体p GEX-4T-1中,获得重组质粒p GEX-VP3。将p GEX-VP3转化BL21感受态细胞后,用1.0mmol/L IPTG于37℃进行诱导。最后,分别用SDS-PAGE和Western-blot对表达产物进行分析和鉴定。结果,NDPV VP3蛋白在大肠杆菌细胞中成功获得表达,表达产物主要以包涵体形式存在,分子质量为84 ku。将纯化的重组VP3蛋白免疫实验家兔,制备多克隆抗体。Western-blot检测结果表明,该抗体能同时与重组的NDPV VP3蛋白及自然感染的NDPV发生反应,说明制备的抗体具有良好的抗原反应性。这些结果为进一步建立NDPV检测方法和研发新型疫苗研究奠定了物质基础。In this research,the VP 3 gene of novel duck parvovirus（NDPV） was amplified by PCR and cloned into the plasmid p GEX-4T-1,then the recombinant plasmid p GEX-VP 3 was transformed into E.coli BL21 for expression induced with 1.0 mmol/L of IPTG. The results of SDS-PAGE and Western-blot showed that NDPV VP 3 gene was expressed successfully in prokaryotic cells.The recombinant VP3 was about 84 ku in molecular weigh.The polyclonal antibody against the NDPV VP3 was prepared with the purified recombinant VP3 through vaccinating rabbits,and it was used to detect the immunogenicity of VP3 in a Western-blot test.The results showed the recombinant protein had a good immunogenicity.Summary,this study laid the good foundation for development of a subunit vaccine and detection methods for NDPV.

关 键 词：新型鸭细小病毒 VP3基因 原核表达 多克隆抗体 

分 类 号：S852.659.2[农业科学—基础兽医学]