新型鸭细小病毒VP3基因克隆与原核表达  

Cloning and Prokaryotic Expression of VP3 Gene of Novel Duck Parvovirus

作　　者：经美 王建昌 崔元[1] 李晓轩 陈萍[1] 王金凤 侯绍华[3] 孙继国[1,4] 袁万哲 JING Mei;WANG Jian-chang;CUI Yuan;LI Xiao-xuan;CHEN Ping;WANG Jin-feng;HOU Shao-hua;SUN Ji-guo;YUAN Wan-zhe(College of Veterinary Medicine,Agriculture University of Hebei,Baoding,Hebei,071001,China;Inspection and Quarantine Technical Center of Hebei Entry-Exit Inspection and Quarantine Bureau,Shijiazhuang,Hebei,050051,China;Beijing Institute of Animal Husbandry and Veterinary Medicine,Chinese Academy of Agricultural Sciences,Beijing,100193,China;Hebei Engineering and Technology Research Center of Veterinary Biotechnology,Baoding,Hebei,071001,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北出入境检验检疫局检验检疫技术中心,河北石家庄050051 [3]中国农业科学院北京畜牧兽医研究所,北京100193 [4]河北省兽医生物技术工程技术研究中心,河北保定071001

出　　处：《动物医学进展》2018年第6期20-23,共4页Progress In Veterinary Medicine

基　　金：河北省高校百名优秀创新人才支持计划(SLRC2017039);国家重点研发计划项目(2017YFD0500806)

摘　　要：应用PCR方法扩增新型鸭细小病毒(NDPV)VP3基因,将其克隆至原核表达载体pQE1中,获得重组质粒pQE1-VP3。重组质粒pQE1-VP3经优化诱导表达后,通过SDS-PAGE对表达产物进行分析并利用His标签抗体对表达的蛋白进行Western blot鉴定。结果表明,NDPV VP3蛋白在25℃、IPTG浓度为1.0mmol/L经诱导5h,可获得最大诱导表达量,表达蛋白的分子质量约为60ku,主要以不溶性包涵体形式存在。纯化复性后,可获得浓度为5.165 mg/mL及纯度为97%的VP3蛋白。获得的重组菌pQE1-VP3以及高纯度的VP3蛋白,为进一步研究NDPV检测方法和新型疫苗奠定了基础。In this research,VP3 gene of novel duck parvovirus（NDPV）was amplified by PCR and cloned into the prokaryotic expression vector pQE1.The recombinant plasmid pQE1-VP3 was optimized in induced expression conditions.The expressed products were analyzed by SDS-PAGE.Results showed that VP3 protein of NDPV was expressed successfully in prokaryotic cells.The protein could obtain maximum expression at 25℃ and 6 hour when the concentration of IPTG was 1.0 mmol/L.The molecular weight of the expressed protein was about 60 ku,mainly existed in the form of insoluble inclusion body.After purification,the protein concentration and purity were respectively 5.165 mg/mL and 97%.In conclusion,VP3 protein of NDPV was efficiently expressed in prokaryotic cells and which laid the foundation for further study on NDPV detection methods and new vaccine.

关 键 词：鸭细小病毒 VP3 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]