非洲马瘟病毒NS1蛋白在昆虫细胞中的表达及抗原性分析  

Expression of African horse sickness virus NS1 protein in insect cells and its antigenicity analysis

作　　者：户鑫兵 王轩莹 宋昱庆 田占成[1] 关贵全[1] 苟惠天[2] 罗建勋[1] 殷宏[1,3] 独军政 HU Xinbing;WANG Xuanying;SONG Yuqing;TIAN Zhancheng;GUAN Guiquan;GOU Huitian;LUO Jianxun;YIN Hong;DU Junzheng(State Key Laboratory for Animal Disease Control and Prevention/College of Veterinary Medicine of Lanzhou University/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China;Gansu Province Research Center for Basic Disciplines of Pathogen Biology,Lanzhou 730046,China)

机构地区：[1]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,兰州大学动物医学与生物安全学院,甘肃兰州730046 [2]甘肃农业大学动物医学院,甘肃兰州730070 [3]江苏高校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 [4]甘肃省病原生物学基础学科研究中心,甘肃兰州730046

出　　处：《中国兽医科学》2024年第3期350-356,共7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);农业科技创新工程项目(CAASASTIP-2016-LVRI)。

摘　　要：非洲马瘟(African horse sickness,AHS)是一种由媒介昆虫传播感染马属动物的病毒性疾病,为了在杆状病毒表达系统中表达AHSVNS1蛋白并评估其作为亚单位疫苗组分的抗原性,本研究参考GenBank上公布的AHSV基因序列,人工合成NS1全长基因序列,利用PCR方法扩增完整的开放阅读框后将其克隆到转座载体pFastBacTMHTb上,将成功构建的重组质粒pFastBac-AHSVNS1转化到DH10Bac感受态细胞中,获得了含有AHSVNS1基因的重组杆粒Bacmid-AHSVNS1,将其转染到Sf9昆虫细胞内获得了表达AHSVNS1蛋白的重组杆状病毒。SDS-PAGE和Western-blot结果显示,表达的重组AHSV NS1蛋白大小为65 ku,且获得了纯化的重组AHSV NS1蛋白。将其免疫BALB/c小鼠,细胞免疫荧光和Western-blot试验显示获得了特异性的抗AHSV NS1蛋白多克隆抗体,其效价达1∶51 200。流式细胞分析显示,AHSV NS1蛋白免疫小鼠能够刺激CD4+和CD8+T淋巴细胞的增加。上述研究结果表明,AHSV NS1蛋白作为AHSV亚单位疫苗的组成成分具有一定应用前景。本试验实现了AHSV NS1蛋白在昆虫细胞中成功表达并验证其具有良好的抗原性,这为进一步研究AHSV NS1蛋白的生物学功能和亚单位疫苗开发奠定了基础。African horse sickness(AHS) is a vector-borne viral disease of equids.To express the AHSV NS1 protein in baculovirus expression system and evaluate its antigenicity as a subunit vaccine component,based on the AHSV gene sequence published in Gen Bank,the full-length NS1 gene of AHSV was artificially synthesized.The complete open reading frame of NS1 gene was amplified by PCR and inserted into vector p Fast BacTMHTb.The recombinant plasmid p Fast Bac-AHSV NS1 was transformed into DH10Bac to obtain recombinant bacmid Bacmid-AHSV NS1.Bacmid-AHSV NS1 was transfected into Sf9 insect cells to rescue the recombinant baculovirus expressing NS1 protein.SDS-PAGE and Western-blot showed that the molecular weight of recombinant AHSV NS1 protein was approximately 65 ku,and the purified recombinant AHSV NS1 protein was obtained.BALB/c mice was immunized with purified AHSV NS1 protein to obtain its polyclonal antibodies.Cellular immunofluorescence assay and Western-blot showed that the specific polyclonal antibodies against AHSV NS1 protein was obtained with a titer of 1 ∶ 52 000.Flow cytometry showed that the mice immunized with recombinant NS1 protein were able to induce the increase of CD4+and CD8+cells,indicating that AHSV NS1 protein has a potential application as a component of AHSV subunit vaccines.This study achieved the successful expression of AHSV NS1 protein in insect cells and verified its antigenicity,which laid a foundation for further investigation on the biological function of AHSV NS1 protein and the development of AHSV subunit vaccine.

关 键 词：非洲马瘟病毒 AHSV NS1蛋白 昆虫细胞表达 抗原性 

分 类 号：S852.659.4[农业科学—基础兽医学]