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AIM:To obtain human recombinant Fv-immunotoxin hscFv_(25)-mTNFα(mutant human TNFαfused to human scFv_(25))againsthepatocellular carcinoma(HCC).METHODS:Two relevant sites of enzymatic digestion wereadded to rnTNFα by PCR.mTNFα was linked to the 3’ endof hscFv_(25)in pGEX4T-1 vector.This anti-HCC recombinantFv-immunotoxin hscFv_(25)-mTNFα was expressed inEscherichia coliand purified from inclusions.After purifiedby glutathione-S-transferase affinity chromatography andthrombin digestion,it was identified by electrophoresis andWestern blot.And then,the purified recombinant Fv-imrnunotoxin was injected into nude mice with HCCxenografts through their tail veins,mTNFα protein and PBSwere used as control at the same time.After treated for twoweeks,nude mice were executed.The bulk and weight oftumors were observed.The tumor tissues were stained byimrnunohistochemical method with TNFα antibody.RESULTS:The expression ratio of recombinant Fv-immunotoxinhscFv_(25)-mTNFα was 12% of bacterial protein.The result oftumor restraining trials of hscFv_(25)-mTNFα showed 2/5 completeremission and 3/5 partial remission,mTNFα restraining trialsshowed 5/5 partial remission.The therapeutic result of hscFv_(25)-mTNFα was better than that of mTNFα(F=8.70,P<0.05).ThehscFv_(25)-mTNFα remedial tumor tissues were positive for TNFαby immunohistochemical staining.The positive granules mainlyexisted in the cytoplasrn of tumor cell.CONCLUSION:Recombinant Fv-immunotoxin hscFv_(25)-mTNFα has better therapeutic effect than mTNFα.It caninhibit the cellular growth of HCC and has some potential ofclinical application.
AIM: To obtain human recombinant Fv-immunotoxin hscFv_ (25) -mTNFα (mutant human TNFαfused to human scFv_ (25)) against hepatocellular carcinoma (HCC). METHODS: Two relevant sites of enzymatic digestion wereadded to rnTNFα by PCR.mTNFα was linked to the 3 ’endof hscFv_ (25) in pGEX4T-1 vector. This anti-HCC recombinant Fv-immunotoxin hscFv_ (25) -mTNFα was expressed in Escherichia coliand purified from inclusions. After purifiedby glutathione-S-transferase affinity chromatography and thrombin digestion, it was identified by electrophoresis and Western blot. And then then, the purified recombinant Fv-imrnunotoxin was injected into nude mice with HCC xenografts through their tail veins, mTNFα protein and PBSwere used as control at the same time. After the treatment for twoweeks, nude mice were executed.The bulk and weight of tumors were observed. The tumor tissue were stained by immunohistochemical method with TNFα antibody .RESULTS: The expression ratio of recombinant Fv-immunotoxinhscFv_ (25) -mTNFα was 12% of bacterial protein.The result of manufacturing restraining trials of hscFv_ (25) -mTNFα showed 2/5 completeremission and 3/5 partial remission, mTNFα restraining trialsshowed 5/5 partial remission. The therapeutic result of hscFv_ (25) -mTNFα was better than that of The hscFv_ (25) -mTNFα remedial tumor tissues were positive for TNFα by immunohistochemical staining. The positive granules mainlyexisted in the cytoplasm of tumor cells. CONCLUSION: Recombinant Fv-immunotoxin hscFv_ (25) -mTNFα has better therapeutic effect than mTNFα. It can inhibit the cellular growth of HCC and has some potential of clinical application.