Identification And Analysis Of Antigen Epitopes Recognized By Monoclonal Antibody Of Avian Leukosis Virus

Avian leukosis?AL?is an infectious tumor disease of chicken caused by avian leukosis virus?ALV?.It is found worldwide and causes immunosuppression,tumors,stunted growth and death in chickens.To date,there is no effective vaccine or drug against the disease.ALV is a retrovirus that is divided into seven subgroups?A/B/C/D/E/J/K?in chickens according to its pathogenicity and antibody neutralization characteristics.Although endogenous ALV-E does not cause serious damage,because its genome has been extensively inserted into host chickens,it interferes with the determination of exogenous ALV.In addition,due to the high variability of exogenous ALV and the continuous emergence of new subgroups and new strains,the detection of exogenous ALV becomes more and more difficult.There is currently a shortage of monoclonal antibodies to detect ALV-A,and little is known about its epitope.In this study,ALV-A monoclonal antibody for detection was prepared and its epitope was identified,providing a scientific basis for the application of monoclonal antibody and a new biological material for the establishment of ALV-A detection method.In this study,gp85 gene was firstly amplified from the whole genome DNA of ALV-A-SDAU09C1 virus by using the primers designed.It was inserted into the pET-32a expression vector.The recombinant plasmid was introduced into E.coli to induce the expression of gp85 protein and then purified.Six-week-aged female Balb/c mice were immunized with purified gp85 protein.After three times of immunization,spleen cells of the mice were fused with SP2/0 cells.Positive hybrid tumor cells were screened by ELISA and IFA,and then cloned and cultured.The monoclonal antibody was determined by ELISA and western blot.Then,the overlapping gp85 protein with 14 truncated segments was expressed and purified in the same way.The complete gp85 protein was used as positive control by western blot to identify the reactivity between the antibody and the gp85 fragment protein and to determine the epitopes of the antigen.Then the synthesis of antigen polypeptide was truncated and the optimum linear epitope was identified by ELISA.Finally,the conformational epitopes and characteristics were analyzed by bioinformatics.Monoclonal antibody mediated IFA was used to analyze the binding effect of monoclonal antibody to different subgroup strains.The results showed that the gp85 gene sequence of 1005 bp was successfully amplified with the designed primers.The size of the gp85 fusion protein induced in E.coli was 46 KD.A positive hybrid tumor cell strain was successfully screened after the fusion of immunized mouse spleen cells and SP2/0 cells,which was named A18GH.The positive hybrid tumor cells were cultured and intraperitoneally injected into mice to prepare ascites.The ascites titer was 1:2¹⁰.Western blot results showed that monoclonal antibody could specifically bind to the prokaryotic gp85 protein and also to the env protein expressed in cells.The ascites were purified by the antibody purification column,and the SDS-PAGE electrophoresis results showed high purity,and the antibody subclass was IgG1.The IFA results showed that monoclonal antibody could bind ALV-A-SDAU09C1 and ALV-K-JS11C1 viruses,but not ALV-B-SDAU09C2 and ALV-J-NX0101 viruses.Then,the reactivity between the fragment expressed gp85 protein,the synthesized peptide and the monoclonal antibody was identified.Western blot and ELISA results of the protein showed that the epitopes of the protein were146-ATRFLLR-152.By analyzing the spatial structure of the antigen epitope,it is concluded that the conformation epitope is a curved linear structure with high hydrophilicity,antigen index,and accessibility.Antigen epitopes were highly conserved in subgroup A,followed by four identical amino acids in subgroup K,and less homologous in other subgroups.The method of IFA was used to identify the ALV subgroup identified by a monoclonal antibody,and the results showed that the monoclonal antibody had a strong reaction with ALV-A-SDAU09C1 strain,a weak reaction with ALV-K-JS11C1 strain,and did not react with ALV-B-SDAU09C2 and ALV-J-NX0101 strains.The above results indicated that this study successfully screened a positive hybrid tumor cell line?A18GH?,which can secrete the anti-ALV-A monoclonal antibody.The epitope recognized by the monoclonal antibody is 146-ATRFLLR-152.