Expression And Characterization Of Recombinant Antibody Against Influenza A Virus In Silkworm Baculovirus Expression Vector System

Influenza A virus threatens human and poultry for a long time,which seriously harms human health and the development of breeding industry.The FI6 antibody involved in this study is derived from human plasma cells.A monoclonal antibody isolated from a large number of human peripheral blood plasma cells was screened by single cell culture method,which can recognize and bind all the serotypes of H1-H16 virus.In this study,the VL segment and VH segment of FI6 were linked by short peptide to constitute the Fv segment.Then the Fv segment was linked to the Fc segment of chicken IgY and human IgG using hinge to obtain two antibodies,namely,chicken-derived antibody(FI6C)and human-derived antibody(FI6H).The amino acid sequence was optimized and kozak sequence was added in order to improve the efficiency of translation initiation,and the stop codon TAA was added to improve the efficiency of protein translation termination.BamHI?EcoR I?Xba I and Pst I sites were added to the two ends of the gene for gene cloning.The target fragment was ligated to the pVL1393 transfer vector by enzyme digestion,and the target fragment was recombined into baculovirus by co-transfection of the transfer vector and the inactivated rescue shuttle plasmid into Bombyx mori cells.Then infected the 5th instar silkworm,harvested silkworm hemolymph,and identified its expression in silkworm by PCR,Western blot and other methods.In vitro experiments were carried out to verify the biological activity of the designed scFv.Hemagglutination inhibition tests showed that FI6H and FI6C had a certain hemagglutination inhibition effect,both of which were 1:160,which was equivalent to hemagglutinin binding ability.The virus neutralization test on chicken fibroblasts showed that the protective effect of FI6H on the cells was better than that of FI6C.The half protective dose of FI6H was pd50=10-4.23/mL silkworm hemolymph.The half protective dose of FI6C was pd50=10-2.79/mL silkworm hemolymph.The results showed that the expression system of Bombyx mori-baculovirus was successfully used to express two neutralizing antibodies of broad-spectrum single-stranded influenza A virus.The biological activity of the neutralizing antibody was tested and showed that it had good biological activity.It lays a foundation for the large-scale production of wide-spectrum recombinant single chain antibody against influenza A virus,and also provides a train of thought for the development of broad-spectrum vaccine.