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目的构建人宫颈癌致癌基因HCCR-1167-360片段的重组质粒,在原核细胞中表达并鉴定。方法从培养的人肝癌细胞株HepG2中提取总RNA,经RT-PCR扩增出HCCR-1167-360的cDNA片段,将其克隆到pGEM-T-Easy克隆载体中,将测序正确的HCCR-1167-360基因连接到pET30a(+)表达载体中,IPTG诱导在BL21中进行表达,用SDS-PAGE检测表达产物。采用初步纯化和割胶纯化方法,获得较纯的重组蛋白,采用弗氏佐剂方法免疫兔子,收获多克隆抗体,用Wastern blot鉴定重组蛋白的免疫原性。结果经测序证明获得了HCCR-1167-360基因片段。SDS-PAGE分析,HCCR-1167-360在BL21中获得表达,其相对分子质量为26kD左右,主要以包涵体形式存在。免疫兔子获得了HCCR-1167-360多克隆抗体,经Wastern blot检测结果表明获得了特异性的多克隆抗体。结论获得具有抗原性的HCCR-1167-360原核表达蛋白。
Objective To construct a recombinant plasmid of human cervical cancer oncogene HCCR-1167-360 and express it in prokaryotic cells. Methods The total RNA was extracted from cultured HepG2 cells. The cDNA of HCCR-1167-360 was amplified by RT-PCR and cloned into pGEM-T-Easy cloning vector. The correct sequence of HCCR-1167 The -360 gene was ligated into the pET30a (+) expression vector, IPTG was induced to express in BL21, and the expression product was detected by SDS-PAGE. Purification and purification methods were used to obtain pure recombinant protein. The rabbit was immunized with Freund’s adjuvant, polyclonal antibody was harvested and the immunogenicity of the recombinant protein was identified by Wastern blot. Results The HCCR-1167-360 gene fragment was confirmed by sequencing. SDS-PAGE analysis, HCCR-1167-360 expression in BL21, the relative molecular mass of about 26kD, mainly in the form of inclusion bodies. Immune rabbit obtained HCCR-1167-360 polyclonal antibody, the results of the Wastern blot showed that specific polyclonal antibodies were obtained. Conclusion The antigenic HCCR-1167-360 prokaryotic expression protein was obtained.