Preparation Of A Single-chain Antibody Fragment Against Highly Pathogenic H5N1 Avian Influenza Virus

Avian influenza (AI) is virolent infectious disease which caused by avian influenza virus A (AIV), AIV mainly infects avian. Three times in the last century, the influenza A viruses have undergone major genetic changes, resulting in global pandemics and large tolls in terms of both disease and deaths. At present, it has been listed as one of A type violently infectious disease by office international des epizooties (OIE). H5N1 began to attack southeastern and central Asia since late 2003. The outbreak of H5N1 virus severely smashered the agricultural economy and led heavy loss of national economy in the outbreaking country. The research of AIV should not be limited to the development of vaccine and diagnostic method. It is should be to pay more attention to therapeutic study of AIV. The situation is improved as the coming of the inhibitors of influenza virus, for example adamantanamine, zanamivir and oseltamivir.Since 1975, Milstein and Kohler have developed hybridoma technique for producting monoclonal antibody (McAb), McAb played an important role in the treatment of diseases nowadays. But the mouse monoclonal antibodies will produce Human Anti-Mouse Antibody, therefore it can't get into the target part of the patients bodies because of its bigger size. Recombinant antibody technology has made possible the design of antibodies that can overcome the limitations of diagnostic and therapeutic monoclonal antibodies. Single chain antibodies (ScFvs) are recombinant antigen-binding molecules that retain the VH and VL domains of whole IgG molecules connected by a short polypeptide linker. They have been shown to have reduced immunogenicity, improved penetration into a solid tumor and have a shorter half life in plasma because of its smaller size.In clinical trials, they are ideal for diagnostic and therapeutic applications.The highly pathogenic strain A/Goose/HLJ/QFY/03 (H5N1 )was chosen as the HA gene donor. The HA gene was cloned into pCI-neo vector to construct recombinant plasmid pCI-HA, that was used as DNA vaccine. BALB/c mice were immunized with pCI-HA for monoclonal antibody preparation. A strain of hybridoma cell, named 3F7, was screened, detected by ELISA and HI test. 3F7 had a high HI titer and a high neutralization. 3F7 was IgG1 subtype and its light chain isκsubtype.A set of degenerate primers were designed for VH and VL clone. And, VH and VL were cloned out from 3F7 cDNA. The sequence of 3F7VH and 3F7VHL were blasted in IMGT/V-QUEST database to identify 3F7 CDRs (complementary determinant region). It was showed that amino acid sequence of 3F7CDRs: VH CDR1: GYTFTETY, CDR2: IYPMNGGT, CDR3: ARKLSLDY; VL CDR1: QSLLNSTNQKNY, CDR2: FAS, CDR3: QQHYSPPPT. 3F7 ScFv was cloned into pET30a to get a prokaryotic recombinant plasmid pET30a-3F7-HL that was constructed by SOE PCR with primer 3F7VH-F and 3F7VL-R. ScFv protein was successfully expressed in E.coli. BL21DE3, when induced with IPTG. The expressed ScFv protein demonstrated HI and virus neutralization activity aganst H5N1 subtype influenza viruses.