结核分枝杆菌rv3295基因的原核表达及单克隆抗体的制备

Prokaryotic expression of rv3295 gene from Mycobacterium tuberculosis and preparation of the monoclonal antibody against the expressed protein

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。

In order to obtain the anti-Rv3295 monoclonal antibody,the rv3295 gene was amplified by PCR using the genomic DNA of MycobacteriumtuberculosisH37 Rv as template,cloned into the expression vector pET-22 bto construct recombinant plasmid pET-22b-Rv3295.This vector was transformed into E.coli BL21(DE3)induced with IPTG and purified by affinity chromatography.After BALB/c mice being immunized with the purified recombinant protein,the anti-Rv3295 monoclonal antibody was prepared by cell fusion and screening.The specificity and subtype of the antibody was identified by Western-blot and the subtype identification kit,respectively.In result,the recombinant Rv3295 protein,with a molecular mass of about 25 ku,was mainly expressed as a soluble form,and a monoclonal antibody 1F7 was obtained.Antibody subtype identification showed that the antibody subtype was IgG1withκchain.Western-blot analysis revealed that the antibody had strong specificity.In conclusion,the prepared monoclonal antibody against Rv3295 protein in the present study laid the foundation for study on the function of Rv3295 protein and the interaction of the protein with DNA.