首页|非洲马瘟病毒NS1蛋白在昆虫细胞中的表达及抗原性分析

非洲马瘟病毒NS1蛋白在昆虫细胞中的表达及抗原性分析

扫码查看
原文链接
  • 国家科技期刊平台
  • NETL
  • NSTL
  • 万方数据
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-AHSV NS1转化到DH10Bac感受态细胞中,获得了含有AHSV NS1基因的重组杆粒Bacmid-AHSV NS1,将其转染到Sf9昆虫细胞内获得了表达AHSV NS1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65ku,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,AHSVNS1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AHSVNS1蛋白的生物学功能和亚单位疫苗开发奠定了基础.
Expression of African horse sickness virus NS1 protein in insect cells and its antigenicity analysis
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 GenBank,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 pFastBacTM HTb.The recombinant plasmid pFastBac-AHSV NS1 was transformed into DH10Bac to obtain recom-binant bacmid Bacmid-AHSV NS1.Bacmid-AHSV NS1 was transfected into Sf9 insect cells to rescue the recom-binant 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.

African horse sickness virusAHSV NS1 proteininsect cell expressionantigenicity

户鑫兵、王轩莹、宋昱庆、田占成、关贵全、苟惠天、罗建勋、殷宏、独军政

展开 >

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

甘肃农业大学动物医学院,甘肃兰州 730070

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

甘肃省病原生物学基础学科研究中心,甘肃兰州 730046

展开 >

非洲马瘟病毒 AHSVNS1蛋白 昆虫细胞表达 抗原性

国家重点研发计划中央级公益性科研院所基本科研业务费专项甘肃省基础研究创新群体项目甘肃省科技厅项目农业科技创新工程项目

2021YFD1800500161031202101422JR5RA02422CX8NA011CAAS-ASTIP-2016-LVRI

2024

10.16656/j.issn.1673-4696.2024.0047

中国兽医科学
中国农业科学院兰州兽医研究所

中国兽医科学

CSTPCD北大核心
影响因子：0.524
ISSN：1673-4696
年,卷(期)：2024.54(3)
  • 18