抗人HBsAg单链抗体基因的构建及其在COS-7细胞中的表达

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(C1y4Ser)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westem blot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。

Construction of single-chain Fv Gene of Anti-HBsAg and its expression in COS-7 cells

AIM:To subclone the V(L) gene and V(H) genes of anti-HBsAg and construct the single-chain Fv gene and to analyse the expression of the constructed gene in COS-7 cells. METHODS: A set of oligonucleotide primers were designed and used to amplify the V(H) and V(L) genes from Fab antibodies screened from phage antibody library. The products were cloned into pUC19 vector and their sequences were analysed. The V(H) and V(L) gene fragments were linked up by a peptide linker and a leader sequence added at 5' terminus of each gene (L-V(H)-Linker-V(L)) and designated as L-ScFv. The L-ScFv genes were inserted into the eukaryotic fusion protein expression vector pEGFP-N3 and transfected into COS-7 cells to express respectively. RESULTS: The coding sequences of V(H),V(L), linker and leader in all constructs were all correct. The expression of ScFv fusion protein was detectable by fluorescence microscope after transient expression in COS-7. The secretive expression of L-ScFv was confirmed by SDS-PAGE and Western blot analysis. And the binding specificity of the ScFvs with HBsAg were proved by indirect ELISA. CONCLUSIONS: Anti-HBsAg ScFv genes have been successfully constructed and expressed in COS-7 cells.