柯萨奇病毒B组5型VP1蛋白的外源表达及多克隆抗体的制备

Prokaryotic expression of coxsackievirus B5 VP1 protein and preparation of polyclonal antibodies

目的:原核表达柯萨奇病毒B组5型的VP1蛋白,并制备其多克隆抗体及检测。方法:提取在Vero细胞中柯萨奇病毒B组5型的RNA,通过逆转录PCR(RT-PCR)扩增获取VP1基因序列,构建原核表达载体,大量诱导表达重组VP1蛋白。用His Trap HP亲和层析柱纯化重组蛋白,以纯化的目的蛋白为抗原,免疫雄性SD大鼠获得VP1蛋白多克隆抗体血清,ELISA测定该抗体的效价,微量中和实验测定抗体的中和活性,Western blot检测抗体的特异性,免疫组化检测抗体对组织中抗原的识别。结果:成功构建了VP1-pET-28a重组载体,并且在BL21细胞中成功诱导表达,亲和层析柱纯化后蛋白纯度大于90%。ELISA测得抗体的效价为1∶128 000,微量中和实验显示抗体没有中和活性,Western blot分析显示抗体能与CVA16和EV71交叉反应,免疫组化实验表明抗体能够识别组织中的CVB5抗原。结论:本研究成功制备了CVB5 VP1蛋白的高效价的多克隆抗体,为CVB5病毒疫苗和病毒诊断的研发提供了有效的工具。

Objective:To express the VP1 protein of Coxsackievirus B5(CVB5)in Prokaryotic cell and prepare its polyclonal antibodies.Methods:The VP1 gene was amplified by reverse transcription-PCR(RT-PCR)from CVB5 grown in Vero cells.A prokaryotic expression vector VP1-pET28a was constructed and transformed into E.coli BL21(DE3).The recombinant protein VP1 was induced by IPTG and then purified with HisTrap FF Affinity Column.The purified target protein was immunized male SD rats to obtain polyclonal antibody against VP1 protein.The titer of the antibody was determined by ELISA.The neutralization activity of the antibody was determinated by microneutralization test.The specificity of the antibody was detected by Western blot and by immunohistochemistry.Results:The recombinant vector VP1-pET28a was successfully constructed.VP1 protein was successfully expressed in BL21 cells.The purity of the protein was greater than 90%after purification by affinity chromatography.The titer of antibody was 1∶128 000 by ELISA.Microneutralization test showed that there was no neutralization activity in antibody.Western blot analysis showed that the antibody could cross-react with CVA16 and EV71.Immunohistochemistry experiments indicated that the antibody could recognize the CVB5 antigen in the tissue.Conclusion:In this study,a high titer polyclonal antibody against CVB5 VP1 protein was successfully prepared,which provided an effective tool for the development of CVB5 vaccine and viral diagnosis.