嵌合口蹄疫病毒VP1抗原基因的重组脑心肌炎病毒的构建及鉴定  

Construction and identification of chimeric foot-and-mouth disease virus VP1 recombinant encephalomyocarditis virus

作　　者：王露露 张武超 雷白时[1,2] 包银莉 王志刚 赵款 袁万哲 WANG Lu-lu;ZHANG Wu-chao;LEI Bai-shi;BAO Yin-li;WANG Zhi-gang;ZHAO Kuan;YUAN Wan-zhe(College of Veterinary Medicine,Hebei Agricultural University,Baoding 071001,Hebei;Hebei Key Laboratory of Analysis and Control of Zoonotic Pathogenic Microorganism,Baoding 071001,China;Longyan University&Fujian Provincial Key Laboratory for the Prevention and Control of Animal Infectious Diseases and Biotechnology,Longyan 364012,China;Duguila Town Comprehensive Support and Technology Extension Center,Ordos 017499,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省兽医生物技术创新中心,河北保定071001 [3]龙岩学院福建省家畜传染病防治与生物技术重点实验室,福建龙岩364012 [4]杭锦旗独贵拉镇综合保障和技术推广中心,内蒙古鄂尔多斯017499

出　　处：《中国兽医科学》2022年第11期1405-1414,共10页Chinese Veterinary Science

基　　金：河北省自然科学基金重点项目(C2019204219);河北农业大学精准畜牧学科群建设项目(1090064);河北省省属高等学校基本科研业务费研究项目(KY2021046);龙岩学院福建省家畜传染病防治与生物技术重点实验室开放基金课题项目(2021ZDKF03)。

摘　　要：将口蹄疫病毒(FMDV)VP1全长、VP1 B细胞表位(135~160、198~213aa)和T细胞表位(20~40aa)组合为不同长度分别插入脑心肌炎病毒(EMCV),构建嵌合FMDV VP1重组EMCV。通过融合PCR及酶切的方法,将外源片段插入EMCV L蛋白第5、6位aa之间,得到重组质粒pWSK-EMCV-FMDV VP1、pWSK-EMCV-FMDV VP1(TB)和pWSK-EMCV-FMDV VP1(2TB),将其转染至BSR T7/5细胞拯救重组病毒EMCV/RvBD_(2)W-VP1、EMCV/RvBD_(2)W-VP1(TB)和EMCV/RvBD_(2)W-VP1(2TB),并在BHK-21细胞传代分析重组病毒的生物学特性。结果显示,PCR扩增得到1344 bp、909 bp和1155 bp的外源基因融合片段,表明成功构建了3种重组质粒;3株重组病毒EMCV/RvBD_(2)W-VP1、EMCV/RvBD_(2)W-VP1(TB)和EMCV/RvBD_(2)W-VP1(2TB)在BHK-21细胞传至F5代,病毒滴度分别为1×10^(7.85)TCID_(50)/mL、1×10^(8.06)TCID_(50)/mL、1×10^(7.76)TCID_(50)/mL;通过RT-PCR、IFA等方法对F5代重组病毒进行鉴定,显示重组病毒EMCV/RvBD_(2)W-VP1(TB)可以稳定遗传,且病毒复制水平与亲本病毒相一致,而其他2株重组病毒在传代过程中缺失。结果表明,本研究构建了3个重组EMCV质粒,转染细胞后携带FMDV VP1抗原表位的重组病毒稳定遗传,为后续以EMCV为骨架构建活载体疫苗奠定了理论基础。Foot-and-mouth disease virus(FMDV)-encoded VP1,VP1 B-cell epitopes(135-160,198-213aa)and T-cell epitopes(20-40aa)were combined into different lengths of fragments and inserted between position 5 and 6 aa of L protein of encephalomyocarditis virus(EMCV),respectively to generate the chimeric plasmids p WSK-FMDV VP1,p WSK-FMDV VP1(TB)and p WSK-FMDV VP1(2TB).After identification by PCR and enzyme digestion,the recombinant plasmids were transfected into BSR T7/5 cells to rescue the recombinant viruses EMCV/Rv BD_(2)W-VP1,EMCV/Rv BD_(2)W-VP1(TB)and EMCV/Rv BD_(2)W-VP1(2TB).The recombinant viruses obtained in BHK-21 cells were passaged and analyzed for biological properties.The results showed that the fusion fragments of 1344 bp,909 bp and 1155 bp were amplified by PCR,indicating that the three recombinant plasmids were successfully constructed.Characterization of the three recombinant viruses EMCV/Rv BD_(2)W-VP1,EMCV/Rv BD_(2)W-VP1(TB)and EMCV/Rv BD_(2)W-VP1(2TB)passed from BHK-21 cells to F5 generation showed,the virus titers were 1×10^(7.85)TCID_(50)/m L,1×10^(8.06)TCID_(50)/m L,1×10^(7.76)TCID_(50)/m L.Further characterization of the recombinant virus by RT-PCR,IFA,demonstarted that the recombinant virus EMCV/Rv BD_(2)W-VP1(TB)can be passaged stably to F5 generations,which was consistent with that of the parental virus.While the other two recombinant viruses failed to passage.In conclusion,three recombinant EMCV plasmids were constructed in this paper.The recombinant viruses carrying the FMDV VP1epitope can be stably passed after transfection into cells,which provided a theoretical basis for the subsequent construction of live vector vaccines with EMCV virus as the backbone.

关 键 词：口蹄疫病毒 脑心肌炎病毒 感染性克隆 VP1 

分 类 号：S852.659.6[农业科学—基础兽医学]