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目的:构建人抗HBsAg单链抗体Tat蛋白转导结构域(ScFvTat)融合基因,在大肠杆菌中进行表达,并分析ScFvTat融合蛋白的活性及内化情况.方法:设计引物扩增人抗HBsAg单链抗体基因,克隆入含有蛋白转导结构域Tat序列的原核表达载体pTATHA,在大肠杆菌BL2l(DE3)LysS内诱导融合蛋白表达.表达产物用SDSPAGE及Westernblot鉴定,用亲和层析柱纯化.间接ELISA和ELISA竞争抑制实验分析ScFvTat融合蛋白的亲和活性,将纯化蛋白与HBsAg阳性或阴性肝癌细胞共孵育后,免疫细胞化学染色分析ScFvTat融合蛋白的结合及内化情况.结果:成功地构建了人抗HBsAg单链抗体Tat融合蛋白的原核表达载体,在IPTG诱导下获得了表达,表达的ScFvTat融合蛋白具有与HBsAg结合的活性,并且能够特异性内化入HBsAg阳性肝癌细胞.结论:单链抗体与Tat蛋白转导结构域融合后,实现了ScFvTat融合蛋白的特异性内化,为该单链抗体的进一步应用奠定了基础.
OBJECTIVE: To construct the ScFvTat fusion gene of human anti-HBsAg single-chain antibody and to express it in E. coli and to analyze the activity and internalization of ScFvTat fusion protein.Methods: Primers were designed to amplify human anti-HBsAg single The recombinant plasmid was cloned into the prokaryotic expression vector pTATHA containing the protein-transducing domain Tat sequence to induce the fusion protein expression in E. coli BL21 (DE3) LysS.The expressed product was identified by SDSPAGE and Western blot and purified by affinity chromatography. Indirect ELISA and ELISA competitive inhibition assay ScFvTat fusion protein affinity activity of the purified protein and HBsAg positive or negative hepatocellular carcinoma cells were incubated with immunocytochemical staining ScFvTat fusion protein binding and internalization.Results: The prokaryotic expression vector of human anti-HBsAg single chain antibody Tat fusion protein was obtained under the induction of IPTG, and the expressed ScFvTat fusion protein has the activity of binding to HBsAg and can specifically internalize into HBsAg positive hepatoma cells.Conclusion: Single After fusion of the chain antibody with the Tat protein transduction domain, the specific internalization of the ScFvTat fusion protein is achieved, and further amplification of the single chain antibody Foundation.