抗肝癌单链免疫毒素在肝癌细胞系SMMC-7721中的表达

EXPRESSION OF ANTI-HCC IMMUNOTOXIN IN HUMAN HCC CELL LINE SMMC-7721.

目的 构建分泌型抗肝癌单链免疫毒素真核表达载体并在人肝癌细胞系 SMMC- 772 1中表达。方法 采用PCR方法在抗肝癌 s Fv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免疫毒素基因 ,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人肝癌细胞系SMMC- 772 1进行瞬时表达。结果 DNA序列分析证实在 s Fv的 5′端引入正确的 6 0 bp引导肽序列 ,酶切鉴定证明成功构建了分泌型抗肝癌单链免疫毒素融合 GFP真核表达载体 ,并在 SMMC- 772 1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免疫毒素融合 GFP基因 。

Objective To study the construction and expression of anti HCC single chain immunotoxin (sFv TNF α) and GFP fusion proteins.Methods The single chain antibody(sFv) gene derived from a monoclonal antibody with high specifity and affinity binding to hepatocellular carcinoma (HCC) was first added leader sequence by polymerase chain reaction(PCR) and then fused inframe with human tumour necrosis factor α(TNF α)gene. The recombinant sFv TNF α gene was cloned into an expression vector pEGFP N3 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, human HCC cell line SMMC 7721 cells were transfected with the plasmid pEGFP ST by Calcium Phosphate method.Results 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 SMMC 7721 cells was observed under the fluorescence microscope 20 hours after transfection of the recombinant vector. Conclusions The gene of single chain immunotoxin was constructed. The fusion proteins of anti HCC single chain immunotoxin and GFP were successfully expressed in SMMC 7721 cells. It is suggested that mammalian cells can express immunotoxin fusion proteins, which may be theoretically necessary for our next step of study in a gene therapy.