人A组轮状病毒G1P[8]基因型VP7蛋白的表达及其抗体制备  

Expression of VP7 protein of group A rotavirus G1P[8]and preparation of antibody to VP7

作　　者：赵颖 苏韫曦 章青[2] 李慧莹[2] 李丹地[2] 李金松[2] 孙晓曼[2] 段招军 Zhao Ying;Su Yunxi;Zhang Qing;Li Huiying;Li Dandi;Li Jinsong;Sun Xiaoman;Duan Zhaojun(School of Public Health,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;NHC Key Laboratory for Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]甘肃中医药大学公共卫生学院,兰州730000 [2]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会医学病毒和病毒病重点实验室,北京102206

出　　处：《中华实验和临床病毒学杂志》2023年第6期643-649,共7页Chinese Journal of Experimental and Clinical Virology

基　　金：国家重点研发计划(2021YFC2301005);国家自然科学基金(32270143,21934005)。

摘　　要：目的表达和纯化人A组G1P[8]轮状病毒VP7蛋白(G1 VP7);制备VP7多克隆抗体并进行抗体功能鉴定。方法利用杆状病毒系统表达G1 VP7蛋白,通过亲和层析法进行蛋白纯化。将纯化后的G1 VP7蛋白免疫新西兰大白兔制备兔多抗血清,纯化得到G1 VP7兔多抗。通过免疫印迹(Western blot,WB)、酶联免疫吸附(ELISA)和免疫荧光试验进行多抗功能验证。结果利用杆状病毒表达系统获得人A组G1P[8]轮状病毒可溶VP7蛋白,并且G1 VP7蛋白主要以三聚体形式存在;制备得到G1 VP7多抗,ELISA结果显示VP7多抗具有较高的效价;WB和ELISA实验表明VP7多抗能够识别多个G型轮状病毒的VP7;免疫荧光试验结果进一步显示G1 VP7多抗可以结合不同A组轮状病毒,包括Wa株(基因型G1P[8])、DS-1(基因型G2P[4])、SA11(基因型G3P[2])、人G9P[8]株。此外,双夹心ELISA初步结果显示包被VP7多抗可以检测到轮状病毒临床样本。结论本研究成功得到可溶G1 VP7蛋白并制备VP7多抗;G1 VP7多抗可以结合多种G型轮状病毒,为不同G型轮状病毒检测方法的建立奠定基础。Objective To express and purify VP7 protein of group A rotavirus(RVA)G1P[8].The VP7 polyclonal antibody was prepared and its function was evaluated.Methods The G1 VP7 protein was expressed by baculovirus expression system and purified by affinity chromatography.Polyclonal antibody against G1 VP7 was obtained by immunizing rabbits with G1 VP7 protein.The function of the G1 VP7 polyclonal antibody was verified by Western blotting(WB),enzyme-linked immunosorbent assay(ELISA),and immunofluorescence assay.Results The soluble G1 VP7 protein of human RVA G1P[8]was obtained using the baculovirus expression system and the VP7 protein was mainly in trimer state.The G1 VP7 polyclonal antibody was prepared and displayed relatively high binding titer to G1 VP7 protein by ELISA.The VP7 polyclonal antibodies could recognize multiple G-type RVAs by WB and ELISA.Immunofluorescence assay further demonstrated that G1 VP7 polyclonal antibody can bind to different RVAs,including Wa(genotype G1P[8]),DS-1(genotype G2P[4]),SA11(genotype G3P[2]),and human G9P[8]RV strains.In addition,double sandwich ELISA showed that VP7 polyclonal antibody could be used to detect rotavirus in clinical samples.Conclusions The soluble G1 VP7 protein was successfully expressed and VP7 antibody was obtained.The G1 VP7 polyclonal antibody could bind to a variety of G-type rotaviruses,which lays a foundation for the establishment of detection method of different G type rotaviruses.

关 键 词：轮状病毒 VP7蛋白 杆状病毒表达系统 多克隆抗体 

分 类 号：R392[医药卫生—免疫学]