The Preparation And Characterization Of Bifunctional Molecule Consisting Of Single Chain Fv Against Human Erythrocyte Fusion HIV-1 Gp41 Antigen Peptide

At present, there were many monoclonal antibodies against human erythrocyte in the world. It had been identified that those antibodies would have widely application if which was modified into the form of ScFv from the apparent antibodies. In these antibodies, IgG was named anti-human-erythrocyte non-agglutinating monoclonal antibodies because this kind of antibodies could bind the red blood cells but not agglutinate erythrocyte in the 0.9% sodium chloride.H antigen was the only antigenical substance in the system of Hb. It distributed on the surface of all human erythrocyte except Bombay type red blood cell. Compared with other specific antigen on the surface of red blood cell, H antigen had advantage on immunological applications because of its broadly distribution.One of the potential immunological application of the monoclonal antibody against H antigen was to establish the detective methods according to the principle of auto-haemagglutinationThe principle of auto-haemagglutination could be described as follow: took a drop of a patient's whole blood and added bifunctional molecules into it. If the antibodies against certain a analyte or the antigen (analyte itself) existed in the blood, the haemagglutination would be observed by naked eyes because of the crosslink of the bifunctional molecules which consisted of two different functional domains, one functional domain could bind erythrocyte and the other could react with the antibodies against the analyte or the antigen (analyte itself).The aim of this study was to establish an auto-haemagglutination detective method that we possessesd the intellectual property for the assay of anti-HIV-1 gp41 antibodies On the basis of hybridoma cell line 2E8 which secret the monoclonal antibodies against H antigen on the surface of human erythrocyte. 1 The clone of variable region gene of 2E8 monoclonal antibodyRT-PCR was used to clone the immunoglobulin (Ig) genes encoding variable regions of heavy and light chains (V_H and V_l) from the hybridoma cell line 2E8, which secrets the monoclonal antibody against H antigen on human erythrocyte. The V_h gene consists of 351bp and the deduced amino acid sequence was closely related to the published sequences of the variable region I (B) subgroup of mouse Ig heavy chain . The V_l gene consists of 339bp and the deduced amino acid sequence was very similar to the published sequences of the variable region I subgroup of mouse Ig kappa light chain.2 The expression and identification of single chain FvThe two variable region genes were spliced by overlap extension and assembled ScFv gene encoding the anti-H antigen, which consists 750bp including the sequence of linker peptide. Then ScFv was transformed into pEGM-T vector and sequenced.The prokaryotic vector pET-his was digested by BamH I and Nhe I enzyme at 37 degree. And the sequenced ScFv gene was cut from pEGM-T vector by the same enzymes and Iigated to pET-his vector with ligase. The recombinant vector was named pET-his-scFv-H.The recombinant vector containing the ScFv gene was transformed into the BL21(DE3) plysS E coli, cells and induced by IPTG. SDS-PAGE and Western blottingdetected a new 32KDa protein band from the BL21 (DE3) plysS cells. The resultof indirect ELISA and competitive ELISA and immunofluorescence indicated that thepurified target protein had the activity of binding the H antigen on red blood cell.3 The expression and identification of single chain Fv against human erythrocyte fusion HIV-1 gp41 antigen peptideOn the basis of sub-cloned single chain Fv gene successfully, the C_H 1 gene from 2E8 MAb and HIV-1gp41 antigen peptide gene was ligated orderly at the V_l terminal of the ScFv gene. The recombinant bifunctional molecules gene consisting of ScFv, C_h1 and HIV-1gp41 gene was 1150 bp. This bifunctional molecule gene was cloned into expression vector pET-his and transformed into BL21 (DE3) plysS E coli, cells and induced by IPTG. SDS-PAGE and Western blotting detected a new 45KDa protein band from the BL21 (DE3) plysS cells. The result of indirect ELISA and competitive ELISA and immunofluorescence indicated that the purified target protein had the activity of binding the H antigen on red blood cell.4 The simulated immunological detective system of the whole blood on thebasis of fusion proteinThe immunological detective system of the whole blood consisted of O type red blood cell, blood plasma containing the antibodies to the HIV-1 gp41. The result indicated that 30 antibody-positive blood plasmas identified by WB could be agglutinated in 5 minutes when the bifunctional molecules were added, and the rate of coincidence was 100%. In 30 samples, 22 were the strong agglutination and 8 were weak agglutination. The haemagglutination was also observed by naked eyes When the bifunctional proteins was added in the simulated immunological detective system of the whole blood consisting of A, B, AB type red blood cells and 6 antibody-positive blood plasmas, respectively. The haemagglutination was not be seen when added bifunctional proteins into the 30 HIV-1 gp41 antibody-negative whole blood (the red blood cell was O type).5 ConclusionOn the basis of 2E8 monoclonal antibody to the human erythrocyte, the bifunctional molecules consisting of the ScFv and HIV-1 gp41 immunogenic peptide were prepared for the detetion of anti-HIV-1gp41 antibodies in the whole blood.