Preparation Of The Monoclonal Antibody Against The Serotype 4 Fowl Adenovirus Fiber2 Protein And Identification Of The Antigenic Epitope

FAdV-4 is a non-enveloped double-stranded DNA virus of the avian adenovirus genus,and one of the important pathogens of HHS.FAdV-4 can be transmitted in poultry through horizontal and vertical routes.FAdV-4 is more and more widely distributed around the world,and poultry of different ages can be infected with FAdV-4,and its fatality rate is as high as 90%,which seriously affects the healthy development of the poultry breeding industry.Therefore,early and rapid detection of FAdV-4 is an effective measure to prevent and control its spread.Studies have shown that the Fiber2 protein located on the surface of FAdV-4 is one of the main virulence proteins of virus infection.It can induce the production of neutralizing antibodies and has a better immune protection effect on HHS.In order to establish a rapid and specific method for detecting FAdV-4,this study first prepared the soluble recombinant protein NusA-Fiber2 using prokaryotic expression technology.The purified recombinant protein NusA-Fiber2 and NusA protein were identified by Western blot.The results showed that NusA-Fiber2 and NusA protein were identified by Western blot.Both Fiber2 protein and NusA protein can specifically bind to His monoclonal antibody,but only NusA-Fiber2 protein can specifically bind to FAdV-4 mouse polyclonal serum.NusA-Fiber2 protein was used as the coating antigen,the coating concentration was 100 ng/mL,and the dilution of the serum to be tested was 1:1280.An indirect ELISA method for detecting antibodies against FAdV-4 Fiber2 with good reaction specificity and simple operation was established.At the same time,the protein was used to prepare 3 monoclonal antibodies against FAdV-4 Fiber2,named 2G5,2G8,and 4C2 respectively.The number of chromosomes of the obtained 2G5,2G8 and 4C2 monoclonal cells was counted,and the average was 104,which is the sum of the chromosome numbers of the two parental cells.Using the mouse monoclonal antibody subclass identification kit to detect the subclasses of the three monoclonal antibodies,the results showed that they are all IgG 1 type antibodies.Protein G was used to purify ascites to obtain MAb 2G5,and its titers were detected by indirect ELISA and IPMA methods.The results showed that their titers were both greater than 1:220 and 1:218.To further verify the specificity of MAb 2G5,LMH cells infected with FAdV-4 were analyzed by IFA and Western blot.IFA test results showed that MAb 2G5 can still have a specific immune reaction with FAdV-4 infected LMH cells at a dilution of 1:500,and green fluorescence can be seen under a fluorescence microscope.However,Mouse IgG doesn’t have a specific immune response to LMH cells infected with FAdV-4.Western blot results showed that MAb 2G5 can still specifically detect FAdV-4 at a dilution of 1:1000.In the neutralization test,the pathological wells were observed under a microscope,and the results showed that MAb can effectively block FAdV-4 infection at the highest dilution of 1:50,while FAdV-4 treated with mouse IgG or without any antibody treatment obvious virus plaques can be seen in the infected LMH cells.MAb 4C2 was used as the capture antibody to coat the ELISA plate,and HRP-2G5 was used as the detection antibody.After a series of conditions were optimized,a double antibody sandwich ELISA detection method for FAdV-4 was established.The established sandwich ELISA method was used to detect cell supernatants infected by IBDV NF8 strain and B87 strain,NDV La Sota strain,Clone30 strain and CS2 strain infected cell supernatant,and the results showed that there was no cross-reaction,indicating that the method has a good specificity.In addition,this method has a minimum detectable concentration of 0.039 μg for Fiber2 protein and 104 TCID50 for FAdV-4,indicating that this method has good sensitivity.The inter-assay and intra-assay coefficient of variation(CV)of this method is less than 10%,which proves that the double antibody sandwich ELISA detection method established in this experiment has good repeatability.By gradually shortening the Fiber2 sequence and using Western blot analysis to identify the epitope sequence range recognized by MAb 2G5 is N-terminal aa.1-aa.33:1MLRAPKRRHSENGKPETEAGPSPAPIKRAKRMV33.In this study,a monoclonal antibody with good reactogenicity was successfully prepared,which laid the foundation for the functional research of Fiber2 protein and the commercial development of a novel FAdV-4 epitope vaccine.