目的通过原核表达系统表达纯化寨卡病毒（zika virus,ZIKV）prM膜蛋白并制备该蛋白的多克隆抗体。方法在对寨卡病毒膜蛋白prM进行生物信息学分析的基础上,去除其跨膜域,对该蛋白的密码子进行优化,化学合成基因序列并连接到表达质粒pET32a,重组质粒转化E.coli.BL21（DE3）,并对其进行测序,然后进行原核诱导表达,诱导产物进行SDS-PAGE鉴定并用His柱纯化。纯化的重组蛋白用于免疫BALB/c雌鼠,制备多克隆抗体,间接ELISA法和Western blot检测血清抗体效价及特异性。结果原核表达系统成功表达了截短prM蛋白,相对分子质量（Mr）为37×10^3,与预期相符。经过His柱纯化后获得高纯度的目的蛋白;该蛋白免疫BALB/c雌鼠后诱导产生特异性多克隆抗体,ELISA效价为1∶819 200;Western blot显示制备的多克隆抗体能性识别prM蛋白。结论利用原核表达系统高效表达并纯化了prM膜蛋白,制备的多克隆抗体效价高,特异性强,为进一步研究该病毒的致病机制及建立ZIKV感染快速诊断方法奠定了基础。
Objectives To express the prM protein of the Zika virus in E.coli,to purify that protein,and to prepare polyclonal antibodies against it. Methods First,the membrane protein prM was analyzed bioinformatically.Then,the codons of the truncated amino acid sequence were optimized for prokaryotic expression in E.coli BL21.The expressed sequence was chemically synthesized,cloned into a pET32 aplasmid,and confirmed as correct with sequencing.The recombinant plasmid was transformed into E.coli BL21 and its expression was induced with IPTG.The recombinant protein was purified using Ni＋affinity chromatography and identified using SDS-PAGE.The purified prM was used to immunize BALB/c mice to prepare a polyclonal antibody.Finally,an indirect ELISA assay and Western blot analysis were performed to determine the titer and specificity of the polyclonal antibody. Results The prokaryotic expression system successfully expressed the truncated prM protein with a relative molecular mass（Mr）of 37×10^3,which was consistent with expectations.The recombinant protein prM was purified using Ni＋affinity chromatography.Indirect ELISA and Western blot analysis indicated that production of a specific polyclonal antibody was induced by immunizing BALB/C female mice with a titer of 1：819 200. Conclusion The prM membrane protein was highly expressed in a prokaryotic expression system and purified using Ni＋affinity chromatography.The prepared polyclonal antibody had a high titer and specificity,thus laying the foundation for further study of the virus＇ pathogenicity and future diagnosis of the Zika virus by detecting the prM protein.
Journal of Pathogen Biology