塞尼卡病毒VP1蛋白的原核表达、纯化及多克隆抗体制备

Expression,purification,and preparation of a polyclonal antibody for the VP1 protein of the Seneca Valley virus

目的利用原核表达系统表达塞尼卡病毒(Seneca Valley virus,SVV)VP1蛋白,并用纯化的蛋白免疫小鼠,制备VP1多克隆抗体。方法PCR扩增SVV VP1蛋白基因,并将其克隆至pET-28a(+)载体,构建pET-28a-VP1重组质粒。将重组质粒转化至BL21(DE3)pLySs大肠埃希菌,使用IPTG诱导表达目的蛋白并优化表达条件,利用His标签镍离子蛋白纯化柱纯化蛋白。用纯化后的蛋白免疫小鼠,制备多克隆抗体,并用接毒后的BHK-21细胞进行Western blot验证。结果PCR扩增SVV VP1目的基因片段为792bp,与预期相符。成功构建pET-28a-VP1重组质粒,转化至DE3后经IPTG诱导表达相对分子质量单位约为35×10^3的目的蛋白,以37℃、0.8mmol/L诱导剂诱导4h表达量最高,且重组蛋白以包涵体形式表达。用His标签Ni^2+-NTA金属螯合蛋白质纯化柱纯化蛋白并免疫小鼠,制备的抗血清能与接毒后的BHK-21细胞裂解产生及纯化的VP1蛋白反应。结论原核表达了塞尼卡病毒VP1蛋白,制备了抗小鼠多克隆抗体,该抗体具有免疫反应性。

Objective To optimize conditions for expression of the VP1 protein of the Seneca Valley virus(SVV)in a prokaryotic expression system and to immunize mice with the purified protein in order to prepare VP1 polyclonal antibodies.Methods The VP1 gene of the SVV was amplified and transfected into a pET-28 a(+)vector,and the recombinant plasmid pET-28 a-VP1 was constructed and transformed into BL21(DE3)pLySs Escherichia coli.Expression of the recombinant VP1 protein was induced with IPTG.The VP1 protein was purified on a His-tag nickel ion protein purification column.The purified protein was used to immunize mice to prepare polyclonal antibodies.Inoculated BHK-21 cells were tested for the antibodies using Western blotting.Results The target gene,VP1 of the SVV,was successfully amplified.The VP1 gene was 792 bp in length,which agreed with expectations.The recombinant plasmid pET-28 a-VP1 was successfully constructed.Expression of the recombinant protein was induced,and the Mr of the target protein about 35×10^3.The level of recombinant protein expression was highest at37℃with 0.8 mmol/L IPTG after 4 h.In addition,the recombinant protein was expressed in the form of inclusion bodies.The protein was purified on a His tag Ni^2+-NTA metal chelating protein purification column.Western blotting was used to detect the response of inoculated BHK-21 cells to the purified target protein.A band of interest was detected at around 35 kDa.Conclusion The VP1 protein of the SVV was expressed in a prokaryotic expression system,and polyclonal antibodies against the VP1 protein were prepared in mice.