二硫键稳定的抗肝癌单链抗体-PE38融合基因的构建、表达与功能检测

目的：制备高特异性有杀伤活性的新型抗肝癌免疫毒素。方法：应用适当的酶切位点，将二硫键稳定的人源化抗肝癌单链抗体(dsFv)基因及PE38基因克隆入原核表达载体pTIG中。诱导表达上清经Ni-NTA亲和层析柱纯化，用MTT比色法检测纯化表达产物的细胞毒活性。结果：目的融合蛋白在大肠杆菌中得到可溶性表达，表达产物的Mr为66000，表达量占菌体总可溶性蛋白量的21％。细胞毒实验表明，抗肝癌免疫毒素对肝癌细胞具有杀伤作用，而对正常肝细胞无损伤。结论：抗肝癌dsFv与PE38融合基因的成功构建及表达，为其作为抗肝癌药物的进一步研究打下了基础。

Construction, expression and functional charactevization of disulfide-stabilized anti-hepatocarcinoma single chain Fv fused with truncated Pseudomonas exotoxin

AIM: To prepare fusion protein of disulfide-stabilized anti-hepatocarcinoma single chain Fv (dsFv) and truncated Pseudomonas exotoxin (PE38). METHODS: The gene encoding the fusion protein was cloned into the prokaryotic expression vector pTIG which was then transformed into E. coli origami (DE3). The expressed product was purified through Ni-NTA agarose affinity chromatography column. The cyto-toxicity of the purified dsFv-PE38 was detected by MTT col-orimetry. RESULTS: The immunotoxin dsFv-PE38 was expressed in E. coli origami (DE3) in a soluble form, which accounted for nearly 21% of the total bacterial soluble proteins. The results of cytotoxic assay showed that the dsFv-PE38 could specifically kill hepatocarcinoma cells, while spared the normal hepatic cells. CONCLUSION: The immunotoxin dsFv-PE38 may have some potential value in the treatment of hepatocarcinoma.