Construction of an immunotoxin HAb25(scFv)-PE40 and its targeting effects against human hepatocellular carcinoma cells / 第三军医大学学报

ABSTRACT

Objective To construct a single chain Fv immunotoxin HAb25(scFv)-PE40 and explore its targeting effects on human hepatocellular carcinoma(HCC) cells.Methods First,the HAb25(scFv) gene was ligated with PE40,a truncated form of pseudomonas exotoxin on the the plasmid vector ply5 to form the HAb25(scFv)-PE40 gene.Then the HAb25(scFv)-PE40 gene was subcloned into the pBV220 E.Coli expression vector for production of the recombinant immunotoxin.SDS-PAGE was used to identify the expression of objective protein.The specificity of HAb25(scFv)-PE40 binding to HCC cells was tested by indirect immunofluorescent staining,and the in vitro targeting cytotoxic assay was employed to observe the selective cytotoxic activity of the objective protein.Results Enzyme digestion and sequencing analysis proved the construction of HAb25(scFv)-PE40 gene in ply5 vector.HAb25(scFv)-PE40 gene was highly expressed in pBV220 vector(up to 43.3% of total cellular proteins).Indirect immunofluorescent staining showed HAb25(scFv)-PE40 could specifically bind to HCC cells as its parent antibody HAb25.The protein was verified to have selective cytotoxic activity when cocultured with HCC cells.Conclusion A single chain Fv immunotoxin HAb25(scFv)-PE40 was successfully constructed.The immunotoxin has selective cytotoxic activity against HCC,which found a basis for further study on its basic and clinical application.