Tamdy病毒糖蛋白基因的重组表达及抗体制备  

Recombinant Expression of Glycoprotein Gene of Tamdy Virus and Antibody Preparation

作　　者：刘利平 沈姝 刘希佳 张敏[1] 王岚[1] 何明宇 孙素荣[1] 张渝疆[3] 丁军涛[1] LIU Liping;SHEN Shu;LIU Xijia;ZHANG Min;WANG Lan;HE Mingyu;SUN Surong;ZHANG Yujiang;DING Juntao(Xinjiang Key Laboratory of Biological Resources and Genetic Engineering,College of Life Science and Technology,Xinjiang University,Urumqi 830046,China;State Key Laboratory of Virology,Wuhan Institute of Virology,Chinese Academy of Sciences,Wuhan 430071,China;Center for Disease Control and Prevention of Xinjiang Uygur Autonomous Region,Urumqi 830002,China)

机构地区：[1]新疆大学、生命科学与技术学院、新疆生物资源基因工程重点实验室,乌鲁木齐830046 [2]中国科学院、武汉病毒研究所、病毒学国家重点实验室,武汉430071 [3]新疆维吾尔自治区疾病预防控制中心,乌鲁木齐830002

出　　处：《病毒学报》2023年第1期13-20,共8页Chinese Journal of Virology

基　　金：国家自然科学基金项目(项目号:81960369,81760365),题目:新型布尼亚样病毒(SFTSV-Like)结构蛋白(SP/GP)线性抗原表位及其自然疫源性研究,准噶尔盆地鼠类和媒介蜱携带重要虫媒病毒病原及其流行病学调查;新疆高校科研计划自然科学重点项目(项目号:XJEDU2019I002);题目:准噶尔盆地新疆出血热自然疫源地调查研究。

摘　　要：原核表达Tamdy病毒(Tamdy virus,TAMV)糖蛋白Gn和Gc,分别制备兔抗融合蛋白Gn和Gc多克隆抗体。利用RT-PCR扩增Gn和Gc基因片段,分别构建原核表达质粒pET-28a-Gn和pET-32a-Gc,并在E.coli BL21(DE3)中诱导表达,镍柱亲和层析纯化融合蛋白Gn和Gc之后,以皮下注射方式分别免疫新西兰兔,制备兔抗融合蛋白Gn和Gc多克隆抗体。经Western Blotting和间接免疫荧光(IFA)鉴定多克隆抗体抗原识别能力,ELISA法测定抗体效价。结果显示,pET-28a-Gn、pET-32a-Gc原核表达质粒构建正确,融合蛋白Gn和Gc大小分别约为45 kD、74 kD,制备的兔抗融合蛋白Gn和Gc多克隆抗体能够分别识别原核表达的融合蛋白Gn和Gc和真核表达产物,效价分别为1∶409 600、1∶204 800。制备的多克隆抗体效价高,能够特异性识别原核系统和真核系统表达的TAMV糖蛋白,为TAMV的流行学分析提供基础。To achieve prokaryotic expression of the glycoproteins Gn and Gc of the Tamdy virus(TAMV), polyclonal antibodies against rabbit anti-fusion proteins Gn and Gc were prepared, respectively. Fragments of the Gn and GC genes were amplified by reverse transcription-polymerase chain reaction(RT-PCR). The prokaryotic expression plasmids pET-28a-Gn and pET-32a-Gc were constructed and induced in Escherichia coli BL21(DE3). The fusion proteins Gn and Gc were purified by nickel-column affinity chromatography. Then, New Zealand rabbits were immunized by subcutaneous injection to prepare a rabbit polyclonal antibody against the fusion proteins Gn and Gc. The antigen-recognition ability of polyclonal antibodies was identified. The prokaryotic expression plasmids of pET-28a-Gn and pET-32a-Gc were constructed correctly, and the size of the fusion proteins Gn and Gc was-45 kD and-74 kD, respectively. The prepared rabbit polyclonal antibodies against fusion proteins Gn and Gc could recognize the prokaryotic-expressed fusion proteins Gn and Gc and eukaryotic expression products with titers of 1:409,600 and 1:204,800, respectively. The polyclonal antibody had a high titer and could specifically recognize the TAMV glycoprotein expressed in the prokaryotic system and eukaryotic system. Our method provides a basis for epidemiological analyses of the TAMV.

关 键 词：Tamdy病毒 糖蛋白 原核表达 真核表达 蛋白纯化 多克隆抗体 

分 类 号：R373.3[医药卫生—病原生物学]