人抗HBsAg单链抗体-Tat融合基因的构建、表达及表达产物的活性鉴定

目的: 构建人抗HBsAg单链抗体-Tat蛋白转导结构域(ScFv-Tat)融合基因,在大肠杆菌中进行表达,并分析ScFv-Tat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pTAT-HA,在大肠杆菌BL2l(DE3)LysS内诱导融合蛋白表达. 表达产物用SDS-PAGE及Western blot鉴定,用亲和层析柱纯化. 间接ELISA和ELISA竞争抑制实验分析ScFv-Tat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ScFv-Tat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ScFv-Tat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ScFv-Tat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.

Gene construction and expression of human anti-HBsAg single-chain Fv-Tat fusion protein in E. coli

AIM: To construct a fusion gene of human anti-HBsAg ScFv-Tat and to analyze the binding activity and internalization of ScFv-Tat fusion protein after its expression in E. coli. METHODS: Two oligonucleotide primers were designed and used to amplify the ScFv gene. The construction was cloned into expression vector pTAT-HA containing the protein transduction domain Tat. The expressed protein was detected by SDS-PAGE and Western blot and purified by affinity chromatography. The binding specificity of the ScFv-Tat fusion protein was confirmed by indirect ELISA and specific internalization of the ScFv-Tat fusion protein was confirmed by immunocytochemistry staining after the purified protein was incubated with HBsAg positive or negative hepatocellular carcinoma cells. RESULTS: Restriction endonuclease digestion and DNA sequencing proved that ScFv gene was correctly cloned into expression vector. SDS-PAGE and Western blot analysis showed that ScFv-Tat fusion protein was successfully expressed in E. coli BL21. Indirect ELISA and competition inhibition ELISA confirmed that the expression products had antigen specific binding activity. ScFv-Tat fusion protein was specifically internalized into HBsAg positive hepatocellular carcinoma cells. CONCLUSION: The anti-HBsAg ScFv-Tat fusion protein expressed in E. coli can specifically internalize into HBsAg positive hepatocellular carcinoma cells.