Preparation And Activity Of Whole Human Intracellular Antibody Against M1 Protein Of H5N1 Avian Influenza Virus

H5N1 highly pathogenic avian influenza is a virulent infectious disease that can be transmitted among birds,and the virus is often carried by wild birds,causing long-distance and wide-scale transnational transmission through migratory birds.At present,24 provinces and autonomous regions in China have issued reports on the H5N1 avian influenza epidemic,which has caused huge direct economic losses,consumed human and material costs,and indirect economic losses to upstream and downstream industries are incalculable.In addition to the harm to the farming industry,the interspecies transmission of H5N1 can also cause great harm to mammals,including humans,and pose a threat to human life and health.In this experiment,antibodies against H5N1 highly pathogenic avian influenza virus were prepared and studied by genetic engineering.Firstly,the TAT protein of human immunodeficiency virus and ScFv,a fully human single-chain antibody gene against M1 protein of H5N1 virus,were fusion-expressed to enable the antibody to cross the cell membrane and gain the ability to bind the virus inside the cell,achieving the effect of preventing the virus from replicating inside the cell.Next,single-molecule antibodies were constructed by fusion expression of a human mFc fragment attached to the antibody to increase its stability and prolong its half-life in vivo.Given that M1 protein is the matrix protein of influenza A virus and is highly conserved among various subtypes of influenza A virus,this experiment was conducted to investigate the broad-spectrum neutralizing effect of intracellular antibody possessing M1 protein against different subtypes of avian influenza viruses.To provide a research basis for further development of broad-spectrum anti-avian influenza virus drugs,the specific results of the study are as follows.（1）pET32（a）-His Tag-SUMO-TAT-ScFv M1-mFc expression vector was successfully constructed,and the stably expressed antibody engineering strain was obtained.Use the combination of SUMO and His Tag,and explore the optimal working conditions of SUMO enzyme.The pure protein with the concentration of 0.18mg/ml was purified by metal chelation chromatography and Pro A affinity chromatography.（2）Using the existing M1 antigen engineering bacteria in the laboratory,the pure M1protein with the concentration of 0.5mg/ml was purified by metal chelation chromatography and cation exchange chromatography as the antigen.And its correctness was identified by Western blot.（3）The TAT-ScFv M1-mFc antibody has strong affinity for M1 protein,and the binding affinity constant is 1.31×10^-8（M）;The detection of antibody activity showed that TAT-ScFv M1-mFc single molecule antibody could inhibit the replication of H1N1 virus when the antibody concentration was greater than or equal to 1.8×10^-4mg/ml,the protective rate against MDCK cells infected by H1N1 virus was more than 95%.Immunofluorescence method was used to verify that TAT-ScFv M1-mFc antibody has model piercing activity;A method for detecting the half-life of TAT-ScFv M1-mFc antibody in mice was established.The maximum half-life of TAT-ScFv M1-mFc antibody was 40 min.