Interaction Between H9N2 Subtype AIV And HSP70 Protein And Development Of Monoclonal Antibody Against M2 Of AIV

Avian influenza virus(AIV)belongs to Orthomyxoviridae of RNA virus,which is divided into three types A,B and C.Influenza virus A is common in birds.,and some influenza viruses A also infect pigs,horses,seals,whales and other mammals and humans.After infected with AIV,some poultry don't show the clinical symptoms,but some poultry cause systemic diseases,with a high mortality rate,even up to 100%.Therefore,according to the pathogenicity,AIVs are divided into low pathogenic avian influenza(LPAI)and high pathogenic avian influenza(HPAI).H9N2 subtype avian influenza virus is one of the most widespread LPAIV in the world,which pose the increasingly serious impact on poultry production and human health,and gradually become the monitoring focuses of influenza virus.However,the functional mechanism of invasion of avian influenza virus into host cells and replication process need to be further explored.In this thesis,HSP70,which interacted with H9N2 subtype AIV in Hela,was screened by virus incubation test and mass spectrometry sequencing analysis.Through overexpression,siRNA and special antibody blocking experiments in HeLa cell,HSP70 protein was verified to might be the receptor protein of AIV when invading into host cells.Furthermore,three truncated HSP70 were constructed to study the functional region of HSP70 protein which play an important role in the invasion of H9N2 subtype AIV.Meanwhile,pET28a-M2 prokaryotic expression vector was successfully constructed,and the recombinant M2 protein was purified to be used as immunogens to successfully prepare the monoclonal antibody against M2 protein of H9N2 AIV.These results provided a good foundation for the research of function of M2 protein and HSP70 protein in H9N2 replication process,and offered the important insight for further development of anti-A influenza virus drugs and research on the pathogenesis and diagnosis technology of H9N2 subtype avian influenza virus.1.Screening and identification of interacting proteins of H9N2 subtype avian influenza virusH9N2 subtype avian influenza virus has become a common influenza A virus in clinical practice,which not only causes catastrophic economic losses to the breeding industry,but also causes serious public health and safety risks.However,the molecular mechanism of H9N2 subtype avian influenza virus invading host cells remains to be further explored.In order to study the key target proteins of H9N2 subtype avian influenza virus for invasion of host cells,in this research,HeLa and A549 cells were used as cell models,and the whole proteins of cells were extracted by ultrasonic method.After PAGE electrophoresis,virus incubation and enzyme-linked antibody detection,the protein bands in HeLa cells and A549 cells were similar,with molecular weight of about 70 KD.The sequence results showed that HSP70 was the binding protein in these both cells that interacted with H9N2 subtype avian influenza virus.To verify the function of HSP70 as receptor protein of avian influenza virus,over expression,siRNA interference and specific antibody blocking were carried out.The results showed that with the expression increase of HSP70 protein,NP protein of AIV also increased in cells.When 1.5?.g/ml HSP70 recombinant plasmid was transfected into cells,mRNA expression of HSP70 was the highest,and the amount of virus mRNA was the highest.However,after 3 ?L siRNA interference,HSP70 protein expression was significantly decreased,whereas NP protein expression was increased slightly,and mRNA level of HA gene was slightly higher than that of control group.Additionally,when HSP70 antibody was used to block the host cells,the expression of HSP70 protein was significantly reduced,and the NP proteins and mRNA levels of HA gene were also reduced.The results suggested that Hsp70 protein might be likely to act as a receptor protein for avian influenza virus to invade host cells,assist virus to invade cells and participate in virus replication process.This study laid a good foundation for further study of the pathogenesis of avian influenza virus and develop the anti-virus targeted drugs.2.Preliminary study on the main functional regions of HSP70 protein involved in the replication of H9N2 subtype AIVHsp70 is an important member of the heat shock protein family.During the replication of influenza A virus,HSP70 can stabilize the viral protein,assist the virus to enter the nucleus and activate the viral replication activity.In order to further explore the functional areas of HSP70 assisting avian influenza virus to invade HeLa cells and virus replication,the structure and biological function regions of HSP70 protein were analyzed,and three truncated fragments of HSP70 gene,namely HSP70-1(24-198aa,525bp),HSP70-2(216-374aa,477bp)and HSP70-3(494-608aa,441bp).Three eukaryotic expression vectors of pEGFP-HSP70-1,pEGFP-HSP70-2 and pEGFP-HSP70-3 were successfully constructed.HeLa cells were transfected with three eukaryotic recombinant plasmids,and then incubated with H9N2 subtype AIV with MOI 1.The effects of three functional regions of HSP70 protein on H9N2 subtype AIV were compared and analyzed by qPCR,Western blot and laser confocal microscopy.The results showed that the expression of viral NP was slightly decreased in the cells transfected with pEGFP-HSP70-1,and the expression of viral NP was not significantly changed in the cells transfected with pEGFP-HSP70-2.Also,it was found that the expression of viral NP was slightly increased,and the mRNA level of viral HA gene was significantly increased in the cells transfected with pEGFP-HSP70-3.Confocal microscopy showed that the NP protein was mainly in the cytoplasm,while the hsp70-3 recombinant protein was mainly in the nucleus.It is suggested that HSP70-3 may be the main functional region of HSP70 protein involved in the invasion and replication of H9N2 subtype AIV.This study provides ideas and methods for further research on the biological function of HSP70 protein,and lays a foundation for the development of specific targeted drugs against avian influenza virus.3.M2 protein expression of H9N2 subtype avian influenza virus and preparation of monoclonal antibodyIt has been confirmed that.HSP70 was involved in the invasion of H9N2 subtype AIV into host cells and virus replication.M2 protein of avian influenza virus is a multifunctional protein,which plays an important role in the invasion,assembly and release of the virus.Therefore,during the replication process of H9N2 subtype AIV,the interaction between M2 protein and HSP70 protein is worthy of further study.In order to study the function of M2 protein,total RNAs of H9N2 subtype avian influenza virus LY1 strain were extracted and M2 gene of H9N2 strain was amplified by PCR.Following gene engineering technology,M2 gene fragment was successfully linked into pET28a prokaryotic expression vector,and recombinant plasmid pET28a-M2 was constructed and expressed.The results of Western blot verified the immunogenicity of the purified fusion protein.Also,the purified virus and M2 protein were used as immunogen to immunize six-week-old female BALB/c mice.Spleen cells of the immunized mice were fused with SP2/0 cells,and purified M2 protein was used as ELISA antigen for detection and subcloning of the hybridoma cells.Results showed that five hybridoma cell lines stably secreting anti-M2 antibody were obtained.The characteristics of these monoclonal antibodies were confirmed by indirect ELISA and indirect immunofluorescence.The results provided a good foundation for further research on the interaction between M2 protein and HSP70 protein in the replication process of H9N2 subtype AIV,and offered the novel insight for further development of anti influenza A drugs and research on the pathogenic mechanism and diagnostic technology of H9N2 subtype AIV.