大口黑鲈弹状病毒G蛋白胞外区原核表达及多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of ExtracellularRegion of G Protein of Micropterus salmoides Rhabdovirus

【目的】获得能特异性识别大口黑鲈弹状病毒(Micropterus salmoides rhabdovirus, MSRV)G蛋白的多克隆抗体,为后续MSRV双抗夹心法胶体金试纸条的研制提供材料。【方法】根据GenBank上MSRV G蛋白的基因序列(GenBank登录号:OK272491.1)设计扩增其胞外区的特异性引物,以感染MSRV的GS细胞的cDNA为模板,通过PCR扩增获得目的序列,双酶切后连接至pET-28a(+)表达载体构建重组质粒pET-28a-ΔG,将重组质粒转化大肠杆菌Trans5α感受态细胞。挑取测序正确的阳性克隆提取重组表达质粒pET-28a-ΔG,转化大肠杆菌BL21(DE3)感受态细胞,再次挑取阳性克隆,测序正确后用IPTG诱导表达。目的蛋白经Ni-NTA树脂层析柱纯化后免疫BALB/c雌性小鼠,4次免疫后眼眶采血获得抗MSRV G蛋白的多克隆抗体,最后通过ELISA、Western blotting和间接免疫荧光试验(IFA)鉴定多克隆抗体的效价和特异性。【结果】PCR结果显示,获得大小为1 323 bp的目的条带。SDS-PAGE结果显示,构建的重组表达质粒可高效表达目的蛋白,Ni柱纯化后获得单一目的蛋白,分子质量在48.5 ku左右,与预期相符,且纯度符合免疫原要求。ELISA结果显示,制备的多克隆抗体效价为1∶204 800。Western blotting结果显示,制备的多克隆抗体可特异性识别不同批次的MSRV G蛋白。IFA结果显示,制备的多克隆抗体能特异性识别天然状态下的G蛋白。【结论】本研究利用MSRV G蛋白的胞外区重组蛋白成功制备出能特异性识别MSRV G蛋白的多克隆抗体,为后续MSRV的免疫检测和疫苗开发奠定了基础。

【Objective】The purpose of this experiment was to obtain polyclonal antibodies that could specifically recognize the G protein of Micropterus salmoides rhabdovirus(MSRV),and provide materials for the subsequent development of the MSRV double antibody sandwich colloidal gold strip.【Method】Based on the genome sequence of MSRV(GenBank No.:OK272491.1),specific primers were designed to amplify the extracellular region of G protein of MSRV.The G gene was amplified via PCR using the cDNA from MSRV infected GS cells,and then ligated into the pET-28a(+)expression vector after double digestion to construct pET-28a-ΔG recombinant plasmid.The recombinant plasmid was transformed into Escherichia coli Trans5αcompetent cells and the positive clone was identified by DNA sequencing.Then,the plasmid pET-28a-ΔG was extracted and transformed into the Escherichia coli BL21(DE3)competent cells.Positive clones were selected again,and the expression was induced by IPTG after correct sequencing.The target protein was purified by Ni-NTA resin chromatography column and used as an immunogen to immunize female BALB/c mice.Polyclonal antibodies against MSRV G protein were obtained by orbital blood collection after four immunizations.Finally,the titer and specificity of the obtained polyclonal antibodies were characterized by ELISA,Western blotting and indirect immunofluorescence assay(IFA).【Result】PCR results showed that a target band with a size of 1323 bp was obtained.SDS-PAGE results showed that the recombinant plasmid could efficiently express the target protein,and a single target protein was obtained after Ni column purification,with a molecular weight of about 48.5 ku,which was consistent with expectations,and the purity met the immunogenic requirements.ELISA results showed that the titer of the prepared polyclonal antibody was 1∶204800.Western blotting results showed that the prepared polyclonal antibodies could specifically recognize different batches of MSRV G proteins.IFA results showed that the prepared polyclonal antibody could specifically recognize G protein in its natural state.【Conclusion】In this study,the recombinant protein of the extracellular region of MSRV G protein was successfully used to prepare polyclonal antibodies that could specifically recognize MSRV G protein,which laid a foundation for subsequent MSRV immune detection and vaccine development.