Cloning And Anti-viral Activity Of ScFvs Against Influenza A Virus

Single-chain antibody fragment(ScFv) is small molecule antibody that prepared bygenetic engineering methods. The recombinant antibody retains heavy chain variable regionand light chain variable region of whole IgG molecules connected by a elastic connectingpeptide. It does not contain fragments of the constant region of the antibody molecule, andhas been shown to have reduced immunogenicity that make antibodies more focused on therole of target cell site.It improved penetration into a solid tumor and have a shorter half Iife inplasma.ScFv has important applications in clinical diagnosis and treatment.A/Goose/Guang dong/1/96(H5N1) and A/Swine/Guangdong/2004(H1N1) was chosen asthe HA gene donor. The HA genes were cloned into pVAX1vector to construct recombinantplasmid pVAX1-HA1and pVAX1-HA5, that was used as DNA vaceines.BALB/c mice wereimmunized with pVAX1-HA1、pVAX1-HA5and inactivated whole virus antigen H1N1、H5N1for monoclonal antibody preparation. Purified GD/04(H1N1) and GD/1/96(H5N1)used as antigen of ELISA reactions to screened positive clones. Finally, we obtained12monoclonal antibodies against H1N1influenza virus and23monoclonal antibody againstH5N1influenza virus. Four strains of anti-H1N1hybridoma cell, named8H1、4D5、5C6and5C3, four strains of anti-H5N1hybridoma cell, named3F2、7H5、3D6�'�3F9were screened,detected by neutralization assay.The total RNA were extracted from the hybridoma cells. VH and VL obtained byRT-PCR and cloned into pMD-18-T vector and sequenced.The sequence of VH and VL wereblasted in IMGT/V-QUEST data base to identify CDRs (complementary determinant region).The heavy chain and light chain genes are connected by a connecting peptide to obtain ScFvgenes with SOE-PCR method. Then, ScFv was cloned into pCDNA-3.1vector to get aeukaryotic recombinant plasmid pCDNA-ScFv.The pCDNA-ScFv transfected MDCK cells to study the activity of anti-influenza virusreplication with virus titration、qPCR、high content imaging system. The results showe that:8H1and4D5can inhibit the replication of H1N1influenza virus significantly, and can inhibitthe spread of the virus.5C6and5C3may be inhibited replication of H1N1influenza virusslightly.3F9can inhibit the replication of H5N1influenza virus significantly, and can reducethe release and spread of the virus.3F2and3D6can inhibit the replication of mild H5N1influenza virus slightly,7H5does not work.The study not only provides support for the anti-viral influenza research and provides anew technique for the prevention of influenza virus.