The Preparation And Identification Of Anti-human AFP ScFv Antibody

Objective: Screening out specific hybridoma cell line who secretes monoclonal antibody can t cross react with human serum albumin (HSA). Separating and purifying VH and VL gene from it and constructing single chain fragment variable (ScFv) gene. ScFv gene was transformed into E.coli and induced it express. Separating and purifying ScFv antibody from Ecoil and identifying it activation. Method: Comparing the homologous sequence of amino acid of human AFP and HSA by GENEDOC software. Identifying the specific of antibody by ELIS A and western blot; Screen out the specific antibody, which can't cross react with HSA. Synthesizing consensus primer according to the sequence of VH and VL of mouse IgG framework region. Extracting the total RNA of hybridoma and separating VH and VL gene by RT-PCR. The ScFv gene was constructed by sequence overlap extend (SOE ) PCR to linked the VH and VL then ScFv gene was cloned into pGME-T vector to construct pGEM-A plasma. pGEM-A was transformed into DH 5 a E.Coli, screen out the white clone and purify the plasma, identify the plasma by endonuclease digest, PCR and sequencing. Digested the pGEM-A and TrxA pET32(a+) vector by EcoR I and Xho I double digest, then purify the ScFv gene which digested from the pGEM-A and legated it into digested pET-32a(+) vector to construct pET-A plasma. CaC12 mediated transformed pET-A plasma into BL-21 (DE3) E.coli.Screening out positive clone by IPTG induces. The plasma was purified and identified by endonucleases digested, PCR and sequencing. The positive E.coli clone was fermented and collected. The germs lyses and purify the inclusion. The inclusion lyses denaturalize in 8M urea and dialyzed renaturate in grads solution to obtain coarse ScFv antibody. Purify the ScFv by enterokinase digested and affinity chromatography, and then identify the ScFv antibody by SDS-PAGE electrophoresis and ELISA competitive inhibited test. Result: There are 2 of 3 line anti-human AFP monoclonal antibodies can react with HSA. The RNA has 28S, 18S &5S three bands, which extracted from the hybridoma. The weight of both of VH and VL, which we obtain by RT-PCR are between 250bp and 500bp.The weight of ScFv about 750bp after VH linked with VL by SOE PCR. The result of endonuclease digestion and PCR show that we have constructed the pGEM-A plasma successfully. The sequencing show that the ScFv gene consisted of 699bp included VH with 339bp and VL with 315bp and linker with 45bp. Obtain 5 positive clone BL-21 (DE3) which express protein after pET-A plasma transformed. The result of endonuclease digestion, PCR& sequencing show that we have constructed pET-A plasma successfully. The ScFv antibody consisted of 233 amino acids, include VH with 113 amino acids and VL with 105 amino acids, there is (G4S)3 linker between the VH and VL.The SDS-PAGE electrophoresis show that we obtain a inclusion with high degree pure after washed with 3M urea and 74% protein have renaturated after dialyzed in grads solution. The molecular weight of TrxA-ScFv fusion protein about 41KD, Scfv about 27KD and the TrxA tag protein about 14KD.The ScFv antibody has high activation tested by ELISA competitive inhibited, the renaturated coarse ScFv can inhibit 25%activation of monoclonal antibody and 37% after it purified. Conclusion: Because of the 50% homogenization of human AFP and HSA.We must exclude the anti-human AFP monoclonal antibody has the cross reaction with HSA. We have constructed the anti-human AFP ScFv gene and express in E.coli with high degree. The ScFv, which we purified from the E.coli, has excellent activation.