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目的构建稳定表达乙肝病毒XTP3蛋白的原核表达载体并在大肠埃希菌中诱导融合蛋白的表达,制备兔抗XTP3蛋白多克隆抗体并检测该抗体在乙肝肝癌、正常肝组织中的作用效果。方法PCR获得XTP3基因片段,插入至pET-32a(+)中构建原核表达载体pET-32a(+)-XTP3,测序正确后转化大肠埃希菌BL21,IPTG诱导表达。SDS-PAGE、Western blotting分析鉴定融合蛋白的表达。利用Ni+亲和柱对表达蛋白进行纯化及柱上复性。免疫新西兰兔,制备多克隆抗体并进行ELISA、Western blotting及免疫组化验证。结果成功构建pET-32a(+)-XTP3表达载体并在大肠埃希菌中诱导出高水平表达的XTP3融合蛋白,目的蛋白分子量为52kD,SDS-PAGE分析表明为包涵体表达。经亲和树脂纯化后免疫新西兰兔,成功获得融合蛋白及兔抗XTP3多克隆抗体。ELISA检测证明多克隆抗体效价>1:128000。免疫组化证实本实验中XTP3多克隆抗体在肝癌组织中的表达呈明显的细胞膜阳性,特异性良好。结论成功表达、纯化了XTP3基因融合蛋白,获得高特异性、高效价兔抗XTP3多克隆抗体,为进一步研究XTP3蛋白的生物学特性奠定了基础。
Objective To construct a prokaryotic expression vector for stable expression of XTP3 protein of hepatitis B virus (HBV) and induce the expression of the fusion protein in Escherichia coli. The anti-XTP3 protein polyclonal antibody was prepared and the effect of the antibody was tested in hepatocellular carcinoma of hepatitis B and normal liver tissues. Methods The XTP3 gene fragment was amplified by PCR and inserted into pET-32a (+) to construct prokaryotic expression vector pET-32a (+) - XTP3. The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG. SDS-PAGE, Western blotting analysis to identify the expression of the fusion protein. The Ni + affinity column was used to purify the expressed protein and renature the column. New Zealand rabbits were immunized and polyclonal antibodies were prepared and tested by ELISA, Western blotting and immunohistochemistry. Results The recombinant plasmid pET-32a (+) - XTP3 was successfully constructed and the highly expressed XTP3 fusion protein was induced in Escherichia coli. The molecular weight of the target protein was 52kD, and the expressed product was expressed by SDS-PAGE. After purified by affinity resin, New Zealand rabbits were immunized and the fusion protein and rabbit anti-XTP3 polyclonal antibody were successfully obtained. ELISA test showed that the polyclonal antibody titer> 1: 128000. Immunohistochemistry confirmed that in this experiment, the expression of XTP3 polyclonal antibody in hepatocellular carcinoma was obvious positive cell membrane, good specificity. Conclusion The XTP3 gene fusion protein was successfully expressed and purified, and a polyclonal antibody against XTP3 with high specificity and high titer was obtained, which laid the foundation for further study on the biological characteristics of XTP3 protein.