非洲猪瘟病毒pp62蛋白在HEK 293F细胞中的表达及其诱导的免疫应答分析  

Expression of African swine fever virus pp62protein in HEK 293Fcells and analysis ofinduced immune response

作　　者：翟文竹 黄颖 陶春豪 何宇恒 储媛媛 王振 庞忠宝 朱鸿飞[1] 贾红[1] ZHAI Wenzhu;HUANG Ying;TAO Chunhao;HE Yuheng;CHU Yuanyuan;WANG Zhen;PANGZhongbao;ZHU Hongfei;JIA Hong(Beijing Institute of Animal Sciences and Veterinary Medicine,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国兽医学报》2024年第12期2514-2520,2555,共8页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2021YFD1801200);云南省重点研发计划资助项目(202103AC100001);农业科技创新计划资助项目(ASTIP-IAS-11)。

摘　　要：非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)引起的高度接触性传染病。为确定ASFV pp62(CP530R)蛋白的免疫原性,本研究利用真核表达重组质粒pMAL-Fc-CP530R转染HEK 293F细胞表达pp62重组蛋白,通过皮下注射免疫C57BL/6J小鼠,10μg/只,分别于0、21d等剂量(加强)免疫1次,通过ELISA、IFN-γELISpot及流式细胞术等方法评价pp62重组蛋白在C57BL/6J小鼠体内的体液免疫及细胞免疫效果。SDS-PAGE结果显示,HEK 293F细胞表达重组蛋白大小约为118.5kDa,与pp62蛋白预期大小一致;Western blot结果显示利用ASFV标准阳性血清可检测到特异性条带;ELISA结果表明,pp62重组蛋白一免后7d即可刺激机体产生特异性抗体,二免后7d抗体效价可达1∶1638400;IFN-γELISpot结果显示,pp62重组蛋白免疫C57BL/6J小鼠可产生IFN-γ,与PBS空白组相比差异显著(P<0.05);流式细胞术检测结果显示,免疫小鼠脾淋巴细胞CD4^(+)/CD8^(+)T细胞亚类比值显著高于PBS空白组(P<0.05);Q-Plex^(TM) Mouse多因子检测结果显示,二免后免疫小鼠血清中IFN-γ、IL-2、IL-4、IL-10分泌水平显著上调(P<0.001)。结果表明,pp62重组蛋白免疫可以激活机体产生较好的体液免疫与细胞免疫,从而为ASFV pp62蛋白功能研究及ASF新型疫苗抗原筛选提供参考。African swine fever(ASF)is a highly contagious disease caused by the African swine fever virus(ASFV).To evaluate the immunogenicity of the pp62(CP530R)protein of ASFV,the recombinant CP530Rprotein was expressed in HEK 293Fcells transfected with the plasmid pMAL-Fc-CP530R.Six-week-old female C57BL/6Jmice were immunized with 10μg of purified pp62protein via subcutaneous injection,followed by a booster immunization with the same dosage at 21days(enhanced).The humoral and cellular immune responses in the mice were then assessed using ELISA,flow cytometry,and ELISpot assays.Western blot analysis confirmed that the pp62 protein was successfully expressed,with a molecular weight of approximately 118.5kDa.ELISA results indicated that a high level of specific antibodies was detected in the immunized mice,with antibody titers reaching up to 1∶1638400at 7days after the secondary immunization.The proportion of CD8^(+)T lymphocytes in the immunized mice increased compared to the control group(P<0.05).Results from the Q-Plex^(TM) Mouse Cytokine Screen demonstrated that the secretion levels of IFN-γ,IL-2,IL-4,and IL-10in serum were significantly upregulated in the immunized mice following secondary immunization(P<0.001).In summary,these findings indicate that the pp62protein can significantly stimulate both humoral and cellular immunity in mice,laying the groundwork for further studies on the function of the ASFV pp62protein and the identification of novel vaccine antigens for ASF.

关 键 词：非洲猪瘟病毒(ASFV) 真核表达 pp62蛋白 免疫应答 

分 类 号：S852.65[农业科学—基础兽医学]