Construction, Expression, Purification And Targeting Study Of Recombinant ScFv25-TNFα And DsFv25-TNFα Against Hepatocellular Carcinoma

1. Expression, purification and targeting study of m/hscFv25 fusing to TNFα against hepatocellular carcinomaMouse anti-hepatocellular carcinoma (HCC) single chain Fv fragment (mscFv25) was fused to human TNFα gene, then mscFv25-TNFα and its humanized counterpart (hscFv25) both were subcloned into prokaryotic GST fusion expression vector pGEX 4T-1, and expressed in the host E.coli JM109 induced by IPTG. Expressed proteins as inclusion bodies were solubilized, refolded and purified by GST affinity chromatography. The specificity and affinity of m/hscFv25-TNFα were not markedly reduced from parental antibody HAb25 for SMMC-7721 antigen, which was confirmed by indirect immunofluorescent staining on target cell. MTT showed that in the presence of actinmycin-D, m/hscFv25-TNFα was cytotoxic to target cell SMMC-7721; if without actinmycin-D, in vitro SMMC-7721 was insensitive to m/hscFv25-TNFα; SMMC-7721 may belong to the TNF-resistant type. In vivo m/hscFv25-TNFα had certain targeting cytotoxicity and caused complete tumor remission in 4 of 14 mice, while no complete regression in TNFα group, suggesting that m/hscFv25-TNFα had certain targeting cytotoxicity, and the cytotoxicity of TNFα must depend on some other factors in vivo. Maybe it damaged the vascular endothelial cell and resulted in ischemic necrosis, or induced the tumor cell to apoptosis, or due to some factors in the role of actinmycin-D or glutathione or transcriptional inhibitors in vivo, but it need further affirmed.