Research Of The Roles Of Linear Neutralizing Epitope To Play In The Immune Protection Of Antibody

The neutralization of an antibody to biotoxin can be decided by its capability of recognizing and binding the neutralizing epitope. So, the neutralizing epitope was taken as the key target of immunoprotection. The linear neutralizing epitope was easy to be mimicked in vitro because of its constant amino acide sequences in primary structure. Thus, this study was conducted to produce human anti-toxin to linear neutralizing epitope by phage display technology and to explore the possibility of preparing antibody by immunizing and scanning with the linear neutralizing epitope.We synthesized epitope peptide G5-24 according to the amino acid sequences of the unique linear neutralizing epitope G5 of rabies virus glycoprotein. The diverse libraries of immunoglobulin heavy and light chain variable gene (VH, VL) were prepared from peripheral blood lymphocytes (PBLs) of donors immunized by G5-24 peptide and vaccine to Rabies virus. The positive scFv phages were selected by four rounds of growth and panning with the G5-24 peptide, and the encoding heavy and light chain genes were sequenced. As a result, we constructed a human phage-scFv library with 8x107 members and obtained an antibody that had correct variable region gene and showen to bind specifically to G5-24 and with good affinities. The antibody's heavy chain and light chain belong to human IgG VH IV family and VK I family respectively. It turned out to be a new scFv gene to rabies virus by the analysis of its CDR.We reconstructed the gene of human scFv (VL-VH) and prepared scFv(VL-VH) and scFv (VH-VL) antibody fragments by prokaryotic expression system PET22b(+)/BL21(DE3). After being purification by Ni-NTA column and renaturalization by urea dialyzation, the affinity and thermostability of scFv(VL-VH) and scFv (VH-VL) were detected. The results suggest we succeeded in constructing scFv (VH-VL) gene and realizing the efficient expressions of scFv(VL-VH) and scFv (VH-VL) in E.coli. And we got active purified recombinatorial proteins which affinitycoefficients were 0.8mol/L and 0.95mol/L respectively. However, their thermostabiiities were not satisfied.We constructed genes of disulfide-stabilized Fv (dsFv) by PCR site mutation that the cysteines were introduced into positon 44 amino acid of VH and position 100 amino acid of VL. The dsFv VH and VL were expressed in PET22b(+)/BL21(DE3) and purified with Ni-NTA column. The results indicate that we constructed dsFv gene and obtained purified human anti-toxin to G5-24.We evaluated the biological activity and protection of dsFv to G5-24 with the contrast to scFv to G5-24. It turned out that dsFv maintained the specific recognization to G5-24 peptide and vero vaccine, and its affinity increased clearly as well as its thermostability and resistantability to urea. dsFv to G5-24 could neutralize rabies virus specifically and inhibit the binding of virus to vero cell, so as to protect the target cell from being infected by rabies virus.Above all, we obtained a specific humanized phage-scFv with higher affinity by immunizing and scanning with linear neutralizing epitope G5-24 peptide. And we succeeded in achieving the stabilization of scFv to G5-24 and obtaining an active human scFv to G5-24. Our study suggested that it is available to get human anti-toxin with the linear neutralizing epitope as immune target.The innovation of the dissertation is to construct a simple, effective method to product human antibody with the linear neutralizing epitope as immune target. And the obtained anti-toxin to G5-24 is a new humanized antibody to rabies virus.