塞尼卡病毒VP1与VP3蛋白原核表达及蛋白纯化  

Prokaryotic expression and purification of VP1or VP3gene of Seneca Valley virus

作　　者：张金勇[1,2] 张赫 孙文超 肖朋朋[3] 南福龙 韩继成 解长占 哈卓 庄忻雨 许汪 鲁会军 金宁一 ZHANG Jin-yong;ZHANG He;SUN Wen-chao;XIAO Peng-peng;NAN Fu-long;HAN Jicheng;XIE Chang-zhan;HA Zhuo;ZHUANG Xin-yu;XU Wang;LU Hui-jun;JIN Ningyi(College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;Military Veterinary Institute,Academy of Military Sciences,Changchun 130122,China;Institute of Virology,Wenzhou University,Wenzhou,Zhejiang 325035,China;College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林农业大学动物科技学院,吉林长春130118 [2]军事科学院军事兽医研究所,吉林长春130122 [3]温州大学病毒学研究所,浙江温州325035 [4]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医学报》2020年第3期463-468,475,共7页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2018YFD0500104)。

摘　　要：利用原核表达系统表达塞尼卡病毒(Seneca Valley virus,SVV)VP1与VP3蛋白,优化其诱导表达条件,并进行蛋白纯化研究。扩增SVV VP1与VP3目的基因,并分别将VP1、VP3克隆至pET32a(+)载体,构建pET32a-VP1与pET32a-VP3重组质粒,将两者分别转化至BL21(DE3)大肠杆菌,经IPTG诱导表达。优化蛋白表达条件,利用His标签镍离子蛋白纯化柱纯化蛋白。RT-PCR结果显示可扩增出792bp SVV VP1与717bp VP3目的片段,将VP1与VP3目的基因成功克隆至pET32a(+)载体,分别转化至BL21(DE3),成功地表达50000pET32a-VP1重组蛋白以及48000pET32a-VP3重组蛋白;pET32a-VP1及pET32a-VP3重组蛋白均在条件为37℃、0.6mmol/L诱导剂诱导5h表达量最高,重组蛋白均以包涵体的形式进行表达并且可纯化出目的蛋白。结果表明,本试验成功地利用原核表达系统表达并纯化SVV VP1与VP3重组蛋白,为制备单克隆与多克隆抗体、检测试剂盒研发以及新型疫苗的研发奠定了基础。To express and purify the VP1or VP3domain of Seneca Valley virus(SVV),prokaryotic expression system were used in this study.The induction conditions were optimized to express a maximum of target protein and then purified target protein.The PCR production of SVV VP1and VP3were cloned to pET32a(+)vector,respectively.Plasmid pET32a-VP1or pET32a-VP3was transformed into BL21(DE3)to express under induction of IPTG,respectively.The induction conditions were optimized to express a maximum of target protein,and the target protein were purified by ProteinIso Ni-IDA Resin.SVV VP1(792bp)or VP3(717bp)gene were amplified by RTPCR,and the target fragments were cloned to pET32a(+)vector successfully.pET32a-VP1or pET32a-VP3recombinant bacteria can express 50000(including labels)or 48000(including labels),respectively.The optimal conditions of pET32a-VP1or pET32a-VP3recombinant bacteria protein expression were 37℃,0.6mmol/L IPTG induced for 5h.Both pET32a-VP1or pET32a-VP3recombinant bacteria were expressed in the form of inclusion body in prokaryotic expression.The pET32a-VP1or pET32a-VP3recombinant protein were purified by ProteinIso Ni-IDA Resin.SVV VP1and VP3recombinant protein were expressed and purified in this research,which laid a foundation of preparation monoclonal antibody or polyclonal antibody,development of a test kit for SVV and research on new vaccines of SVV.

关 键 词：塞尼卡病毒 VP1 VP3 原核表达 蛋白纯化 

分 类 号：S852.65[农业科学—基础兽医学]