ABSTRACT
Objective:To construct anti-B7-H4-scFv-PE38KDEL,a recombinant toxin based on anti-B7-H4 single chain antibody (scFv),to detect anti-tumor effect of toxin protein.Methods:The anti-B7-H4-scFv gene was ligated with the toxin PE38KDEL gene by overlapping extension PCR(SOE-PCR).The recombinant gene was cloned into prokaryotic expression vector pET28a(+),and the protein was renatured and purified by chromatography (Ni-NTA),and was identified by Western blot.Indirect ELISA and flow analysis technology were used for specific identification.The inhibitory effects of toxins on tumor cells were detected by MTT assay and subcutaneous xenograft model in vitro and in vivo.HE staining and immunohistochemical analysis were performed on tumor tissues.Results:The recombinant expression vector pET28a-anti-B7-H4-scFv-PE38KDEL was obtained by restriction endonuclease di-gestion.The purified toxin protein was inoculated on the tumor cells.The tumor growth was inhibited in the tumor model.Conclusion:The recombinant toxin expression system based on anti-B7-H4 single chain antibody was successfully constructed.The recombinant toxin protein had good biological activity and anti-tumor activity.
ABSTRACT
Objective To construct a recombinant immunotoxin expression vector composed of a single-chain Fv fragment of Sehistosorna japomicum and PE38KDEL gene,and identify the binding activity of the purified product with SEA antigen.Methods The V_H and V_L genes were amplified by PCR from the parent monoclonal antibody NP11-4.Then the amplified scFv and PE38KDEL genes were inserted into the expression vector pBAD/gIII A.The fusion protein expressed in E.coli Top10F' induced by L-arabinose.After purification,the activity of the immunotoxin was evaluated by Westem-blot and ELISA.Results The new recombinant immunotoxin expression vector pBAD/gIII A-scfv-PE38KDEL was constructed successfully.The main product was in inclusion bodies.ELISA assay showed that the refolding recombinant immunotoxin remained binding activity with SEA antigen.Conclusion A new recombinant expression plasmid pBAD/gIII A-scfv-PE38KDEL has been constructed and expressed successfully,which is useful in further study of the treatment of schistosomiasis japonica.
ABSTRACT
Objective: To observe the targeting therapy of the hdsFv-RC-RNase recombinant single chain immunotoxin on the xenograft of the human hepatocellular carcinoma in nude mice and to explore its clinical potentiality. Methods: The prokaryotic expression vector TIG-hdsFv-RC-RNase was transformed into E.coli BL21(DE3)plys to largely express recombinant single chain immunotoxin hdsFv-RC-RNase against hepatocellular carcinoma induced by IPTG. The expressed product was purified via Ni-NTA affinity chromatography under native conditions and mildly refolded. The ELISA was used to analyze its immunological activity of antigen-binding capability. The xenogrft model of the human hepatocellular carcinoma in nude mice was established and the targeting therapy of hdsFv-RC-RNase was evaluated. Results:After induced by IPTG, a new protein band with M_r 41 000 was found in the supernatant of the bacteria and expressed in a soluble form. The expressed product was purified to homogeneity via Ni-NTA affinity chromatography under native conditions. The results of ELISA showed the refolded hdsFv-RC-RNase had the specific antigen-binding capability to the human hepatocellular carcinoma cell, but not to the normal hepatocyte (P