Preparation Of Monoclonal Antibodies Against HA And NP Protein Of H9N2 Subtype Avian Influenza Virus And Its Preliminary Application In Antigen Variation Analysis

H9N2 subtype avian influenza virus(AIV)circulate widely in the word since its first detection.In China,H9N2 subtype AIV is endemic and spreads widely,which not only causes huge economic losses to the poultry industry,but also poses a potential threat to human beings.Under the influence of immune pressure and antigen drift,the antigenic variation of H9N2 subtype AIV often happen,which resulted in the failure of the vaccine immunization in the end.Therefore,it is of great significance to develop an ideal McAb against H9N2 subtype AIV for monitoring and diagnosis of H9 subtype AIV1.Cloning and expression of HA and NP gene of H9N2 AIVThe H9N2 subtype AIV epidemic strain A/Chicken/Taixing/10/2010(rTX)and vaccine strain A/Chicken/Shanghai/F/98(F98)were selected as target strains.Their HA genes and NP genes were amplified by PCR and cloned into the eukaryotic expression vector PCAGGS.The recombinant plasmids PCAGGS-rTX-HA,PCAGGS-F98-HA and PCAGGS-rTX-NP were confirmed to be constructed successfully.Next,these plasmids were transfected into 293T cells respectively.Through indirect immunofluorescence assay(IFA)and Western blot(WB)test,it was confirmed that HA and NP proteins were correctly expressed in vitro,and the immunogen for monoclonal antibody(McAb)preparation was successfully prepared.Meanwhile,to construct recombinant plasmid pET32a-rTX-NP,NP gene was sub-cloned to a prokaryotic expression vector pET-32a.Roestta(DE3)transformed with the recombinant plasmid was induced by IPTG and the soluble NP was expressed effectively with molecular weight of 73 KD.The rNP was obtained as detecion antigen for the next preparation of monoclonal antibody against NP protein of rTX strain.2.Preparation of monoclonal antibodies against H9N2 subtype AIV HA protein and its primary application in antigen variation analysisBALB/c mice were immunized with plasmids PCAGGS-rTX-HA and PCAGGS-F98-HA by intramuscular injection and gene transfection apparatus.After the third immunization,inactivated virus was injected intraperitoneally to enhance the immunity.Spleen cell of immunized mice were fused with SP2/0 cell after three days.Hemagglutination inhibition test(HI)and IFA are used to screen HA-positive hybridoma cells.After three subclones,6 McAbs were obtained.Four McAbs against the rTX HA protein were named 1B9,1C10,3E2 and 2D6,Two McAbs against the F98 HA protein were named 1B2 and 1F10.The subclass identification results of antibody showed the subclasses of 1B9,1C10 and 3E2 were IgM,the subclass of 2D6 was IgG2a,and the 1B2 and 1F10were IgG1.The HI titers of 6 McAbs ascites were higher,all among 210-215.The result of IFA showed that 1B9,1C10,3E2 and 2D6 had specific reactoin to rTX,while 1B2 and 1F10 had specific reaction to F98.Western-blot assay showed that 1B2 and 1F10 had specific reaction with strain F98,and the remaining McAbs had specific reaction to strain rTX.The neutralization test showed that 2D6,1B2 and IF 10 all had neutralizing properties,of which 1F10 had the highest neutralizing titer.The results of specificity and broad-spectrum of these McAbs showed that the six McAbs against HA protein only reacted with H9 subtype AIV,but not with other subtypes AIV and NDV,indicating that they had good specificity.Four of them had better binding spectrum to H9N2 AIV.The results of HI and IFA detection of six McAbs against the isolated strains in 2017-2019 showed that 37.1%(10/27)of H9N2 AIV isolates had mutant antigenic epitopes differentiated from Y280-like strains,and 22.6%(6/27)of H9N2 AIV isolates got smiliar antigenic epitopes as F98-like strains.Therefore,these McAbs can used a tool for antigenic variation analysis,and there is obvious antigenic variation in H9N2 AIVs isolated in 2017-2019.3.Preparation and identification of monoclonal antibodies against H9N2 subtype AIV NP proteinRecombinant plasmid PCAGGS-rTX-NP was injected into mice to prepare McAbs.After 10 days of cell fusion,positive hybridoma cells were screened by Enzyme-linked immunosorbent assay(ELISA)and IFA.After 3 subclones,6 McAbs were finally obtained,named 1B4,1E50,2E10,3A9,8F10 and 3E6.The subclass identification results of McAbs showed the subclasses of 2E10,3A9 and 1B4 were IgG2a,the subclass of 1E50 was IgGl,and the subclasses of 3E6 and 8F10 were IgM.The titers of McAb ascites were detected by ELISA test,the titers were among 1:25600-1:204800.The results of IFA showed that only 3E6 and 8F10 had specific fluorescence reaction with rTX strain.The specificity results showed that the 3E6,8F10,2E10 and 1B4 did not react with NDV and IBV,and had good specificity.While the specificities of 3A9 and 1E50 were not very good,which also could react with NDV.The results of broad-spectrum showed that 3E6,8F10 and 2E10 had good broad-spectrum reaction with H9N2,H5N6 and H7 subtypes AIV,but did not have reaction with H5N1 subtypes.While 1B4,3A9 and 1E50 had poor broad-spectrum response only with H9N2 subtype AIV and the strain named W1-8(H7 subtype).In summary,3E6,8F10 and 2E10 have better comprehensive characteristics,which have certain application value in the epidemiological detection and early diagnosis of AIV.