非洲马瘟病毒重组VP7蛋白在大肠杆菌中的表达及多克隆抗体的制备与鉴定  

Expression of recombinant African horse sickness virus VP7 protein inEscherichia coli and preparation and identification of its polyclonal antibodies

作　　者：王轩莹 户鑫兵 罗昭依 田占成[2] 高闪电[2] 殷宏[2,3] 罗建勋 关贵全[2] 苟惠天[1] 独军政[2] WANG Xuan-ying;HU Xin-bing;LUO Zhao-yi;TIAN Zhan-cheng;GAO Shan-dian;YIN Hong;LUO Jian-xun;GUAN Gui-quan;GOU Hui-tian;DU Jun-zheng(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory for Animal Disease Control and Prevention/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室,甘肃兰州730046 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2023年第8期971-976,共6页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2021YFD1800500);中央级公益性科研院所基本科研业务费项目(1610312021014);甘肃省基础研究创新群体项目(22JR5RA024);甘肃省科技厅项目(22CX8NA011);农业科技创新工程(CAAS-ASTIP-2016-LVRI)。

摘　　要：非洲马瘟是由非洲马瘟病毒感染马引起的一种以发热、皮下和肺组织水肿及内脏出血为主要特征的烈性传染病。本研究首先通过PCR以合成的AHSV S7基因为模板扩增得到开放阅读框片段,将其克隆至大肠杆菌表达载体pPROEX HTb,成功构建重组质粒pPROEX-AHSVS7。转化重组质粒pPROEX-AHS-VS7到大肠杆菌BL21(DE3)感受态细胞中,用0.5 mmol/L IPTG进行诱导表达,通过SDS-PAGE和免疫印迹(Western-blot)对表达产物进行鉴定。采用包涵体和亲和层析纯化的方法对表达产物进行纯化,获得纯度较高的重组VP7蛋白,经3次免疫新西兰大白兔收集血清并进行抗体纯化,通过Western-blot、免疫荧光和间接ELISA法进行抗体的鉴定。结果表明,本试验利用大肠杆菌系统成功诱导表达了约为40 ku的重组AHSV VP7蛋白,并成功制备出特异的VP7多克隆抗体。以上结果为进一步研究VP7蛋白的生物学功能、建立诊断方法及亚单位疫苗研发提供了基础。African horse sickness is a virulent infectious disease caused by African horse sickness virus,which is characterised by fever,subcutaneous and pulmonary oedema and internal haemorrhage.In this study,we first obtained the open reading frame fragment of AHSV S7 gene by PCR amplifica-tion and then cloned it into the E.coli expression vector pPROEX HTb to construct the recombinant plasmid pPROEX-AHSVS7.The correctly sequenced recombinant plasmids were transformed into E.coli BL21(DE3),which was induced with 0.5 mmol/L IPTG and the expression of the target protein was identified by SDS-PAGE and Western-blot.The purified recombinant VP7 protein was obtained by Ni-chelating affinity chromatography.Anti-VP7 sera were collected from New Zealand rabbits by three immunizations and antibody purification was performed,and the specificity of the antibody was verified by Western-blot,immunofluorescence and indirect ELISA.The results showed that the recombinant AHSV VP7 protein was successfully expressed using the E.coli system,and the specific polyclonal antibodies against VP7 protein were prepared,which provides a basis for further research on the biological function of VP7 protein,the establishment of diagnostic methods and the development of subunit vaccines of African horse sickness.

关 键 词：非洲马瘟病毒 VP7蛋白 多克隆抗体 

分 类 号：S852.659.4[农业科学—基础兽医学]