分泌型抗肝癌单链双功能抗体融合GFP真核表达载体的构建及表达

Construction and expression of anti-HCC immunotoxin of sFv-TNF-α and GFP fusion proteins

目的分泌型抗肝癌单链双功能抗体融合GFP真核表达载体的构建及表达。方法采用PCR方法在抗肝癌sFv的5端引入引导序列使其能够在真核细胞中表达并分泌,在其下游连接人TNF-α基因,构建分泌型抗肝癌单链双功能抗体基因,并将该基因克隆入带有GFP报告基因的真核表达载体,用磷酸钙共沉淀法转染小鼠成纤维细胞NIH3T3进行瞬时表达。结果 DNA序列分析证实在sFv的5端引入正确的60bp引导肽序列,酶切鉴定证明成功构建了分泌型抗肝癌单链双功能抗体融合GFP真核表达载体,并在NIH3T3细胞中成功地表达了融合荧光蛋白。结论成功构建并表达了分泌型抗肝癌单链双功能抗体融合GFP基因,为肝癌免疫基因治疗的进一步研究奠定了基础。

AIM To study the construction and expression of antiHCC single-chain bifunctional antibody (sFv-TNF-α) and GFP fusion proteins. METHODS A single-chain antibody (sFv) gene comprising the linked heavy and light chain variable regions clerived from a monoclonal antibody with high speciflty and affinity binding to hepatocellular carcinoma (HCC) were first added leader sequence by polymerase chain reaction (PCR) and then fused inframe with human tumor necrosis factor-α (TNF-α) gene. The recombinant sFv-TNF-α gene was cloned into an expression vector pEGFP-N_3 containing the report gene of GFP, and the recombinant vector was first identified with enzyme digestions for the correct Insertion and further confirmed by DNA sequence analysis. To investigate whether mammalian cells could produce fusion protein, NIH3T3 cells were transfected with the plasmid pEGFP-ST by Caiclum Phosphate method. PESULTS The recombinant vector was identified with enzyme digestions for the correct insertion and further confirmed by DNA sequence analysis. Bright green fluorescence In the cytoplasm of the transfected NIH3T3 cells was observed under the fluorescence microscope 20 hours after transfectlon of the recombinant vector. CONCLUSION The gene of single-chain bifunctional antibody was constructed. The fusion proteins of anti-HCC single-chain bifunctional antibody and GFP were successfully expressed in NIH-3T3 calls. It is suggested that mammalian cells can express antibody-cytokine fusion proteins, which may be theoretically necessary for our next step of study in a gene therapy.