人源性抗HBc单链抗体真核表达载体的构建及其细胞内表达

Construction of human anti-HBc ScFv eukaryotic expression vector and expression of anti-HBc ScFv in HepG2 cells

目的构建人源性抗HBc单链抗体的真核表达载体并在细胞内表达。方法采用DNA重组技术将特异性人源性抗HBc单链抗体基因插入真核表达载体pEGFP-c1;转染HepG2细胞,经G418筛选细胞,荧光倒置显微镜观察细胞内抗HBc单链抗体与绿色荧光蛋白融合表达情况,并用ELISA法检测HBc单链抗体基因的细胞内表达。结果成功地构建了人源性抗HBc单链抗体的真核表达载体。转染HepG2细胞并筛选后,经荧光倒置显微镜观察,细胞内有绿色荧光蛋白表达;ELISA检测细胞内表达的单链抗体片段具有HBcAg结合活性。结论人源性抗HBc单链抗体的真核表达载体的构建并在细胞内成功表达,为胞内抗HBc单链抗体的进一步研究奠定了基础。

AIM: To construct an eukaryotic expression vector of human single-chain variable fragment ~against hepatitis B virus core protein (anti-HBc ScFv) and detect its expression in HepG2 cells. METHODS: Anti-HBc ScFv genes were amplified from the plasmids abstracted from positive clone and inserted into pEGFP-c1 vector that contained green fluorescent protein gene. The recombinant plasmids were transfected into HepG2 cells, and resistant clones were obtained by G418 selection. The expression of the gene of fusion protein was determined by fluorescent invert microscope and ELISA. RESULTS: Recombinant plasmids were successfully constructed. The plasmid transfected HepG2 cells were obtained by G418 selection. Specific fluorescence was observed in HepG2 cells 48 hours after transfection. ELISA analysis confirmed the expression of anti-HBc ScFv in the cells. CONCLUSION: The construction of human anti-HBc ScFv eukaryotic expression vector and its expression in HepG2 cells lay the foundation for advanced research of intracellular anti-HBc ScFv.