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以结核分枝杆菌H37Rv标准株基因组DNA为模板,通过PCR扩增了rv3295基因;将其克隆至表达载体pET-22b中,构建了重组质粒pET-22b-Rv3295;将重组质粒转化到大肠杆菌BL21(DE3)中,经IPTG诱导表达出了Rv3295蛋白。通过亲和层析纯化,以该重组蛋白为免疫原免疫BALB/c小鼠,利用细胞融合筛选制备抗该重组蛋白的单克隆抗体;采用Western-blot验证单克隆抗体的特异性;通过亚型鉴定试剂盒鉴定单克隆抗体的亚型。结果显示,Rv3295蛋白以可溶形式表达,蛋白质的分子质量大小为25ku。获得1株抗该重组蛋白的单克隆抗体1F7,其亚型为IgG1/κ;Western-blot结果显示,该抗体具有较好的特异性。本研究制备的Rv3295蛋白的单克隆抗体对于研究Rv3295蛋白的功能及其与DNA之间的互作奠定了基础。
The rv3295 gene was amplified by PCR using genomic DNA of Mycobacterium tuberculosis H37Rv as a template. The recombinant plasmid pET-22b-Rv3295 was constructed by cloning it into the expression vector pET-22b. The recombinant plasmid was transformed into E. coli BL21 (DE3), induced by IPTG expression of Rv3295 protein. Purified by affinity chromatography, immunized BALB / c mice with the recombinant protein as an immunogen, and the monoclonal antibody against the recombinant protein was prepared by cell fusion screening; the specificity of the monoclonal antibody was verified by Western-blot; Identification kits identify subtypes of monoclonal antibodies. The results showed that Rv3295 protein was expressed in soluble form, the molecular mass of protein was 25ku. Obtained a monoclonal antibody against the recombinant protein 1F7, the subtype of IgG1 / κ; Western-blot results showed that the antibody has good specificity. The monoclonal antibody of Rv3295 protein prepared in this study laid the foundation for studying the function of Rv3295 protein and its interaction with DNA.