Construction and expression of disulfide-stabilized humanized scFv and hEDN recombinant immunotoxin fusion gene against hepatocellular carcinoma

Objective:To study the construction and expression of a novel recombinant immunotoxin fusion gene prokaryotic expression vector composed of disulfide-stabilized humanized scFv (hdsFv) and hEDN gene against hepatocellular carcinoma (HCC) in E.coli. [WT5HZ]Methods: PCR was used to amplify the hEDN gene fragment encoding the mature protein. Recombinant immunotoxin fusion gene of hdsFv- hEDN prokaryotic expression vector was constructed by linkage hdsFv gene against HCC with hEDN gene and introduced into E.coli cell BL21(DE3)plyS. The expression of recombinant immunotoxin hdsFv-hEDN against HCC was induced by IPTG in E.coli. The expression product was analyzed and identified by SDS-PAGE and Western-blot.[WT5HZ] Results:A 470 bp long gene fragment was amplified by PCR, and the size and sequence were accorded with those of hEDN reported in Genebank. PCR identification and nucleotide sequencing confirmed that the construction of recombinant immunotoxin fusion gene prokaryotic expression vector was correct. After induced by IPTG, the recombinant immunotoxin hdsFv-hEDN against HCC was successfully expressed in E.coli. A new anticipated Mr 45 KD protein band appeared on SDS-PAGE gel and amounted to 22% of total bacterial protein. The expressed product existed in a form of inclusion body that was confirmed by Western-blot. [WT5HZ]Conclusion:The prokaryotic expression vector of recombinant immunotoxin fusion gene hdsFv-hEDN against HCC is successfully constructed and induced to express recombinant immunotoxin, which lay the foundation for the further study of targeting therapy of HCC. [WT5HZ]