The Selection Of Human Monoclonal Fab Fragments Against HIV-1 Gp120 Peptide Binding Chemokine Receptor From Phage Fab Antibody Library

mRNA temples were extracted from the peripheral blood lymphocytes with antibodies against human immunodeficiency virus 1 type(HIV-1) donated by persons who infected HIV-1. Two pairs of primers were synthesized based on the nueleotide sequences of human immunoglobulin heavy chain Fd fragment and κ chain respectively. The heavy chain Fd fragment and K chain were amplified by RT-PCR respectively taking the extracted mRNA as temples, inserted into the pComb3 vector. The vector was transformed into Escherichia coli TG1 by electroporing. Phage Fab antibody library with capicity of 1O~13CFU/ml was constructed. A synthesized polypeptide containing 23 amino acid residues including Arg-419, Ile-42O, Lys-421, Gln-422, Pro-438 of the gpl20 antigen epitope binding chemokine receptor was coated as the solid-phase antigen. After 6 cycles of biopanning(absorption,elution and amplification) and ELISA and dot blot 7 strains of huamn phage Fab monoclonal antibody against HIV-1 gpl20 polypeptide were acquired. The results of inhibition test showed that the inhibition rates3of the 7 strains of Fab monoclonal antibody to HIV-1 antibody-positive serum are 68. 18% to 79. 38%. The results of the competitive inhibition rate of the Fab monoclonal antibody No. 67 against HIV-1 anbody-positive serum is about 50%. It showed the similar binding power with gpl20 polypeptide between Fab monoclonal antibody No. 67 and HIV-l antibody-positive serum. The results of specific inhibition test showed that only the gpl20 polypeptide we synthesized can inhibit the binding between the 7 strains of Fab monoelonal antibody with solid-phase antigen.We extracted recombinant phagemids of the 4 clones (No. 7, No.67, No.76, No. 81)chosed from all the 7 clones. The DNA fragments about 2. 5kb including Fd fragment and K gene were gotten from each of the 4 clones digested by Xho I and Xba I . Fd fragments about 700 bp were gotten from clones No. 7 and No. 67 digested by Xho I and Spe I. K genes about 650bp were gotten from clones No. 7 and No. 67 digested by Sac I and Xba I . Fd fragments about 700 bp were ampilified by PCR from clones No. 76 and No. 81. So does the K gene about 650bp and the DNA fragment about 2. 5kb including Fd fragment and K gene. DNA sequencing of the 4 clones showed the identical sequences of Fd fragment and K gene . The sequences we got were compared with the sequences in the GenBank. The Fd fragment belongs to IgG I subclass and the light chain belongs to K type. The VH and V were derived from VN IIIand VAIl.This study suggested that the acquired human phage Fab monoclonal antibody against HIV-1 gpl20 polypeptide has good specificity and high affinity. It can compete with HIV-1 antibody-positive serum to bind gpl20 antigen site binding chemokine receptor. This provided potential promise to get4the genetic engineer pharmacon which can blocks the HIV-1 infection efficiently.