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Objective: To Obtain purified genetic eogineering recombinant scFv-fusion protein with potentialities of clinical application. Methods: Mouse anti-hepatocellular carcinoma (HCC) single chain Fv fragment (mscFv25 ) was fused to human TNFa gene, then mscFv25-TNFa was subcloned into prokaryotic GST fusion expression vector pGEX 4T-l, and ex-. pressed in the host E. colt induced by IPTG. Expressed proteins as inclusion bodies were solubilized, solubilized and purified by GST affinity chromatography. The cytotoxity of mscFv25-TNFa was evaluated on SMMC-7721 by MTT, and the targeting therapeutic value was revealed in nude mice bearing HCC xenografts. Results: The specificity and affinity of mscFv25-TNFa were not markedly reduced compared with its parental antibody HAb25 against SMMC-7721 antigen. In vitro target cell SMMC-7721 was insensitive to mscFv25-TNFa. In the mscFv25-TNFa had certain targeting cytotoxicity and caused complete tumor disappearance in 4 of 14 mice, and the side effects of TNFa were much weaker in mscFv25-TNFa group than in group. Conclusion: SMMC-7721 may belong to the TNF-resistant type. While in the trial, mscFv25-TNFa caused complete tumor disappearance in 4 of 14 mice, but no disappearance in TNFa group, suggesting that mscFv25-TNFa had certain tar geting cytotoxicity, and the cytotoxicity of TNFa depended on some other factors in the. It may damage vascular endothelial cell and lead to ischemic necrosis, or induce the tumor cell to apoptosis and some agents to play the the of actinmycin-D in vivo. The targeting of mscFv25 can diminish unspecific cytotoxicity of TNFa, thus attenuate the side effects of TNFa.
Objective: To obtain purified genetic eogineering recombinant scFv-fusion protein with potentialities of clinical application. Methods: Mouse anti-hepatocellular carcinoma (HCC) single chain Fv fragment (mscFv25) was fused to human TNFa gene, then mscFv25-TNFa was subcloned into prokaryotic GST fusion expression vector pGEX 4T-l, and ex-. pressed in the host E. colt induced by IPTG. Expressed proteins as inclusion bodies were solubilized, solubilized and purified by GST affinity chromatography. The cytotoxity of mscFv25-TNFa was evaluated on SMMC. -7721 by MTT, and the targeting therapeutic value was revealed in nude mice bearing HCC xenografts. Results: The specificity and affinity of mscFv25-TNFa were not markedly reduced compared with its parental antibody HAb25 against SMMC-7721 antigen. In vitro target cell SMMC -7721 was insensitive to mscFv25-TNFa. In the mscFv25-TNFa had certain targeting cytotoxicity and caused complete tumor disappearance in 4 of 14 mice, and the side effects Of TNFa were much weaker in mscFv25-TNFa group than in group. Conclusive: SMMC-7721 may belong to the TNF-resistant type. While in the trial, mscFv25-TNFa caused complete tumor disappearance in 4 of 14 mice, but no disappearance in TNFa group, suggesting that mscFv25-TNFa had certain tar geting cytotoxicity, and the cytotoxicity of TNFa depended on some other factors in the. It may damage vascular endothelial cell and lead to ischemic necrosis, or induce the tumor cell to apoptosis and some agents to Play the the actinmycin-D in vivo. The targeting of mscFv25 can reduce unspecific cytotoxicity of TNFa, thus attenuate the side effects of TNFa.