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目的 :构建抗人HBsAg单链抗体基因 ,并分析其在COS 7细胞中的表达。方法 :以从噬菌体抗体库中筛选的抗HBsAg的Fab抗体基因为模板 ,分别扩增其轻、重链可变区 (VL、VH)基因 ,通过重组PCR方法将轻、重链可变区基因用连接肽(Gly4Ser) 3的编码序列连接 ,并引入前导肽编码序列 ,构建具有L VH Linker VL 结构的单链抗体基因。将所构建的单链抗体基因克隆入绿色荧光蛋白 (GFP)融合表达载体pEGFP N3,并转染COS 7细胞进行表达。结果 :经测序表明 ,前导肽、连接肽、VL 及VH 的序列正确。酶切鉴定证实 ,成功地构建了GFP基因融合表达载体。瞬时转染COS 7细胞后 ,通过荧光显微镜观察证实有ScFv融合蛋白的表达。转染细胞的培养上清浓缩后 ,进行SDS PAGE及Westernblot分析 ,可检出ScFv融合蛋白的分泌性表达。培养上清的间接ELISA检测证实 ,所表达的单链抗体具有与HBsAg结合的特异性。结论 :成功地构建了抗人HBsAg单链抗体基因 ,并可在COS 7细胞中分泌性表达
Objective: To construct the anti-human HBsAg single-chain antibody gene and analyze its expression in COS-7 cells. Methods: The light and heavy chain variable region (VL, VH) genes were amplified by using the anti-HBsAg Fab antibody gene selected from the phage antibody library as a template. The light and heavy chain variable region genes Linking with the coding sequence of the peptide (Gly4Ser) 3 and introducing the leader peptide coding sequence, a single-chain antibody gene with the L VH Linker VL structure was constructed. The constructed single-chain antibody gene was cloned into green fluorescent protein (GFP) fusion expression vector pEGFP N3 and transfected into COS 7 cells for expression. Results: Sequencing showed that the leader peptides, linker peptides, VL and VH sequences were correct. Enzyme digestion confirmed that the GFP gene fusion expression vector was successfully constructed. After transient transfection of COS 7 cells, the expression of the ScFv fusion protein was confirmed by fluorescence microscopy. The supernatant of the transfected cells was concentrated and analyzed by SDS PAGE and Western blot to detect the secreted expression of the ScFv fusion protein. Indirect ELISA detection of the culture supernatant confirmed that the expressed single chain antibody has the specificity of binding to HBsAg. Conclusion: The anti-human HBsAg single chain antibody gene was successfully constructed and secreted in COS 7 cells