Preparation And Activity Reseach Of The H3N2 Canine Influenza Virus M1 Protein Intracellular Antibody

Canine influenza virus?CIV?belongs to the genus of influenza A virus of the Orthomyxovirus family,and its clinical symptoms include high fever,shortness of breath,purulent nasal discharge,anorexia,depression and hemorrhagic pneumonia.At present,epidemiological studies have confirmed that equine H3N8 and avian H3N2subtype canine influenza virus can spread stably in canines.The successful implementation of influenza virus from equine to canine,from avian to canine,and canine gradually become A"hotbed"of evolution and reproduction of influenza viruses.As a companion animal of human beings,canines have a special status in modern human life,but the potential risk of canines as an intermediate host of influenza virus and transmitting the virus to humans will pose a great threat to human health.Because influenza viruses are prone to antigenic drift and antigen transfer,neutralizing antibodies and vaccines against viral surface antigens need to be constantly updated,and the timeliness of vaccines has great problems.Therefore,research on universal canine influenza virus antibody drugs is urgent.In this study,the highly conserved M1 protein of influenza virus was used as a target,the specific scFv was screened in the phage antibody library,and the immunological binding activity and biological activity of scFv were studied.Then,TAT protein with high transmembrane transduction function was used to construct TAT-scFv shuttle intracellular antibody,and evaluated the effect of intracellular antibodies on the proliferation of three heterologous influenza viruses?A/Canine/Gua-angdong/05/2011?H3N2?,A/Duck/Guangdong/W12/2011?H3N2?,A/Beijing/1/1968?H3N2??at the cellular level and in the SPF chicken embryo model.1.Prokaryotic expression and identification of H3N2 canine influenza virus M1proteinIn order to prepare pure protein of canine influenza virus?H3N2?M1 protein,primers were designed for the highly conserved sequence of H3N2 canine influenza virus M1 protein,and the target gene fragment was amplified by PCR.The amplified product was cloned into expression vector pET-SUMO and transformed into host strain BL21?DE3?,The M1 gene of 771bp,which is amplified by PCR,successfully constructed the pET-SUMO-M1 expression plasmid.After purification by enzyme digestion,the pure M1 protein was obtained,which provided a pure antigen for further preparation of the universal anti-H3N2 canine influenza virus antibody.2.Screening and activity study of anti-H3N2 canine influenza virus M1 protein scFvUsing M1 as a target protein,a single-chain antibody against M1 protein was screened from the phage antibody library Tomlinson I library?library capacity:1.47×10⁸?and Tomlinson J library?library capacity:1.37×10⁸?by stationary phase screening.After four rounds of biopanning,it was shown that the enrichment factor for high affinity positive phage clones was increased for each additional round of screening.The positive strains were screened by indirect ELISA and PCR,and the immunological binding activity of C2 strain and M1 target protein was the most significant.The antibody gene of C2 strain was analyzed and had complete single-chain antibody structure.The C2 strain was expanded and successfully purified as H3N2-scFv.The protein concentration of BCA was 1.6 mg/mL,and the purity of the thin layer was 98.9%.The purified scFv was identified by WB method,and results showed that the prepared target protein of scFv and M1 has good immunological binding activity.Biological activity studies at the cellular level and in the SPF chicken embryo model showed that scFv had no significant toxic effects on cells and chicken embryos at concentrations up to 1.6mg/mL,and significantly suppressed A/Canine/G-uangdong/05/2011?H3N2?canine influenza virus replication in a dose-dependent manner.The cell level IC₅₀=0.290mg/mL and the chicken embryo level IC₅₀=0.120mg/mL were measured,which proved that the sensitivity of scFv was higher in chicken embryos.3.Preparation and activity of TAT-scFv recombinant shuttle intracellular antibodyThe C2 strain pIT2-scFv plasmid was used as a template for PCR amplification,and the 867bp scFv gene fragment was successfully obtained.The amplified product was cloned into the expression vector pET-28?a?-TAT to construct the recombinant expression plasmid pET-28?a?-TAT-scFv.PCR and double enzyme digestion showed that the inserted fragment size was 867bp,which proved that the pET-28?a?-TAT-scFv recombinant plasmid was successfully constructed.The recombinant plasmid was transformed into the host strain BL21?DE3?to induce the expression of the target protein.After 12%SDS-PAGE analysis,there was a band at the size of 32kDa,which proved that the recombinant protein of TAT-scFv was successfully expressed.The expression of recombinant protein was the highest when the expression bacteria were cultured in TB medium,and induced at 30°C for 6h under 0.6mmol/L IPTG conditions.After the expression culture was subjected to pilot fermentation and expanded culture,the recombinant protein of TAT-scFv with a concentration of2.0mg/mL was purified to a purity of 93.655%.The purified TAT-scFv was identified by WB method,and the results showed that it appeared at a size of 32kDa.A specific immunological band demonstrated successful preparation of TAT-scFv shuttle intracellular antibodies.The difference in immunocom bination activity between TAT-scFv and scFv and M1 protein was detected by indirect ELISA,and the results showed that both dilutions from 1:10 to 1:640 had good immunological binding activity with M1 protein,and there was no significant difference,and the TAT cell transmembrane protein did not affect the biological activity of the scFv single chain antibody.Genetic evolution analysis of three H3N2 influenza viruses showed that instead of belonging to a same evolutionary branch,the three H3N2 influenza viruses belong to canines,birds or humans.Three heterologous H3N2 influenza viruses affected MDCK cells and SPF chicken embryos respectively,and the effects of different concentrations of TAT-scFv on the proliferation of influenza virus were studied.Toxicity studies showed that TAT-scFv had no significant toxicity to cells and chicken embryos at concentrations up to 1.6mg/mL,and cell viability was still 94.1%,and chicken embryos were all normal,and blood vessels showed no dead embryos,and no other pathology changes.The change indicates that the protein itself is not toxic to cells and chicken embryos.Bioactivity studies showed that TAT-scFv significantly inhibited the proliferation of three H3N2 influenza viruses in both MDCK cells and chicken embryos.It also showed a dose-effect relationship.The effect of TAT-scFv on the inhibition of proliferation of three H3N2 influenza viruses:the inhibition of A/Canine/Guangdong/05/2011?H3N2?and A/Duck/Guangdong/W12/2011?H3N2?was higher than that of A/Beijing/1/1968?H3N2?,which indicates that the inhibition of TAT-scFv on the proliferation of three H3N2 influenza viruses was related to the branching of the virus during evolution.The TAT-scFv shuttle intracellular antibody prevents the assembly and release of influenza virus by disrupting the M1 matrix protein of the H3N2 influenza virus.By observing the survival rate of chicken embryos,the evaluation of TAT-scFv antiviral drugs was completed,and it was found that 1.6 mg/mL of TAT-scFv in 8 days could achieve 100%protection against chicken embryos infected with 100uLEID₅₀ of A/Canine/Guangdong/05/2011?H3N2?and A/Duck/Guangdong/W12/2011?H3N2?,for infection 100uLEID₅₀ the A/Beijing/1/1968?H3N2?chicken embryo group can achieve 50%protection.In this study,H3N2 canine influenza virus M1 protein was successfully prepared,and scFv was successfully screened with M1 protein as antigen.TAT protein was used as transmembrane transporter to successfully prepare TAT-scFv shuttle intracellular antibody.The study showed that TAT-scFv shuttle intracellular antibody was different.The proliferation of the H3N2 subtype influenza virus has an inhibitory effect,which indicates that TAT-scFv has the potential to become a universal anti-H3N2 subtype influenza virus drug.