| Hepatocellular carcinoma(HCC), namely hepatocarcinoma, is one of the most popular and fatal malignant tumors. It has a hideous latency, early translocation and a high incidence fatality which make it have a name of "the king of cancer". It is very difficult to cure the hepatocarcinoma now and the surgery therapy, chemotherapy and actiontherapy are still the main methods. But the targeted therapy based on antibodies make the cure of cancer promising, there are many targeted agents being developed for cancer therapy with single-chain Fv, disulfide-stabilized Fv or Fab fragment as targeting moieties and cytokines, toxins, superantigens or radionucides as bullets.Among theses targeting moieties, disulfide-stabilized Fv or Fab fragment has disulfide bond to conjoin the heavy chain and the light chain to increase the stablility, but it is difficult to express in host or has a poor yield. Single-chain Fv has mature prokaryocyte expression system with high yield, but its stability is poor. To circumvent this problem, we construct the sinle-chain disulfide-stabilized Fv fragment targeted superantgen SEA(D227A) molecule to increase its yield and stability. Single-chain disulfide-stabilized Fv fragment is new developed type of Fv fragment based on single-chain Fv by site-directed mutagenesis to cysteine in the framework region of heavy chain and light chain to create an interdomain disulfide bond in the Fv and increase the yield and stability.In this research, we constructed the single-chain disulfide-stabilized Fv antibody, this promising Fv form, by connecting the VH and VL of anti-hepatoma disulfide-stabilized Fv antibody with linker, and then constructed the recombinant expression plasmid of scdsFv-SEA(D227A) by fusing the scdsFv antibody with superantigen SEA(D227A).The expression plasmid of scdsFv-SEA(D227A) was induced to express as inclusion bodies in E.coli BL21plusS by IPTG and the yield of inclusion bodies is 30% of the total bacterial protein. The inclusion bodies were renatured after washed many times and denatured. And then, the solution was purified by Q Sepharose HP and Hiprep 26/60 Sephacryl S-200 HR. The purity of the recombinant protein was upon to 90% and the yield of the purified protein was 60 mg per liter of induced culture.AMS alkylation and PAGE electrophoresis analysis showed that intramolecular disulfide bond formed correctly in the recombinant protein.ELISA and MTS assay showed that the purified protein had similar binding affinity as dsFv fused SEA(D227A) and scFv fused SEA(D227A) have and similar killing activity as native SEA has to human hepatoma cell line.But ELISA assay shows that the stability of scdsFv-SEA(D227A) is improved greatly. It can be stable for at least nine days of exposure in PBS and surprsingly it remains more than 50% binding activity even after exposure to temperature at 56 °C for 30 min or incubation with 5M urea for 30 min at 37 °C which show that recombinant protein has the anti-denaturant stability activity and thermal stability activity.In conclusion, this research establishes a novel method to stabilize the immunotoxins and increase their yields. And the strategy makes our anti-hepatoma hScFv25 antibody targeted superantigen to apply promising in clinic. Therefore, we propose that this strategy can be applied to improve stability and yield of unstable/low yield Fv-immunotoxins.
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