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

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.