脆性组氨酸三联体基因与HIV-TAT蛋白转导域融合蛋白表达、纯化和多克隆抗体制备

Studies on expression and purification of FHIT/TAT fusion protein and preparation of its polyclonal antibody

目的:制备脆性组氨酸三联体(FHIT)基因与HIV-TAT蛋白转导域的融合蛋白FHIT/TAT以及该融合蛋白的兔抗多克隆抗体,探讨FHIT基因对肝肿瘤的作用及其机制.方法:构建FHIT基因与HIV-TAT蛋白转导域的融合蛋白表达载体,确定该载体的最佳诱导表达条件,在大肠杆菌中大量表达并获得纯化的FHIT/TAT融合蛋白,同时以该融合蛋白免疫兔制备抗FHIT/TAT融合蛋白的多克隆抗体.结果:构建了FHIT基因与HIV-TAT蛋白转导域的融合蛋白表达载体;确定了该载体的最佳诱导表达条件;Western Blot检测也证实了该融合蛋白在大肠杆菌中的表达;以此为基础在大肠杆菌中大量表达并获得了70%纯度的FHIT/TAT融合蛋白;同时制备了较高滴度的兔抗FHIT/TAT融合蛋白多克隆抗体.结论:成功建立了制备具有高效转导作用的HIV-TAT蛋白转导域与FHIT基因融合蛋白的原核表达体系,获得了该融合蛋白的兔抗多克隆抗体,为FHIT基因对于肝肿瘤的作用及其机制研究,特别是肝肿瘤的基因治疗和预防等打下了良好的基础.

AIM： To prepare fusion protein of fragile histidine triad （FHIT） gene with HIV-TAT protein transduction domain and to acquire rabbit polyclonal antibody against the fusion protein so as to lay a good foundation for further research on the function and mechanism of FHIT gene in hepatocellular carcinoma. METHODS： Prokaryotic expression vector of FHIT gene was constructed with HIV-TAT protein transduction domain and its optimal expression conditions were investigated. FHIT/TAT fusion protein was acquired by E. coli expression system and subsequent purification process and rabbits were immuned with the purified fusion protein to acquire polyclonal antibody against it. RESULTS： The expression vector of FHIT/TAT fusion protein was successfully constructed and the optimal expression conditions were determined. The target protein＇s accurate expression was confirmed by Western Blot analysis. The purity of FHIT/TAT fusion protein was nearly 70% and higher titer rabbit polyclonal antibody against FHIT/TAT fusion protein was acquired. CONCLUSION： Prokaryotic expression system of FHIT/TAT fusion protein with high transduction efficiency has been successfully established and higher titer rabbit polyclonal antibody against FHIT/TAT fusion protein has been acquired, which lays a good foundation for the function and mechanism research of FHIT gene on hepatocellular carcinoma, especially on the gene therapy and prevention of hepatocellular carcinoma.