Expression Of Subgroup K Avian Leukosis Virus Gp85 Gene Specific Fragment And Its Application

K subgroup avian leukemia virus(ALV-K)is a newly discovered subgroup of exogenous avian leukemia virus in recent years.This subgroup mainly exists in Chinese local chickens.The virus proliferates slowly and has a wide range of infections,which is one of the main targets for prevention and control of local chicken flocks.Compared with other subgroups of avian leukemia viruses,the research on this subgroup of viruses started late,the infection characteristics and pathogenic mechanisms of this virus are less known,and specific diagnostic reagents and methods are lacking,which has greatly restricted the in-depth study,clinical prevention and control testing of this virus.The purpose of this study is to based on the genetic characteristics of the envelope surface protein of the ALV-K strain and use molecular biology,cell biology and veterinary immunology and other technical methods to obtain specific protein antigens and antibodies against this virus subgroup,then establish antigen-antibody-mediated specific detection methods which can provide a material basis and methodological basis for in-depth research and clinical prevention and control of this subgroup of viruses.In this study,based on the ALV-K envelope surface protein gp85 gene sequence published on Gen Bank,we use bioinformatics software to screen out specific antigen gene fragments with low homology to other subgroups and design primers.This study uses the gene of the ALV-K-JS11C1 strain as a template to amplify the gene fragment,and uses prokaryotic expression technology to obtain a specific protein antigen;secondly,this study purifies,identifies,coats the protein antigen and optimizes the reaction conditions to establish an ELISA method for detecting antibodies to this subgroup of viruses,then evaluates the specificity,sensitivity,repeatability,coincidence rate and storage life of the method;third,this study uses the protein antigen to immunize rabbits,prepares polyclonal antibodies,then detects their antibody titer and the recognition characteristics of virus or antigen;fourth,we use the protein antigen to immunize Bal b/c mice to prepare antibody-secreting splenocytes,hybridize and fusion with SP2/0 cells to obtain antibody-secreting hybridoma cells.And this study tests the number of chromosomes,types of secreted antibodies and recognition characteristics of tumor cells.The results showed that the size of the specific antigen gene fragment was 310 bp,and the homology with ALV-K subgroups published so far was 92.9%?100%,and the homology with other subgroups was 37%?79.7%;the phylogenetic tree shows that this gene fragment is closely related to the ALV-K subgroup strain gene,and is farther from other subgroups;the protein structure has high antigenicity.After gene clone and induced expression,a target protein band with a size of 30kDa was obtained.We purify and coat the protein to establish an ELISA,and optimize the antigen coating concentration,antibody dilution,reaction conditions,etc.,this study determines the optimal conditions that the antigen coating concentration is0.75?g/m L,the serum antibody dilution is 1:800,and the secondary antibody dilution concentration is 1:5000,the time for primary antibody incubation is 45min,the time for secondary antibody incubation is 60min,and the time of developing color is 10min.This ELISA can distinguish ALV-K from other ALV-A/B/J subgroups of chicken serum antibodies;the sensitivity of this method is nearly 20 times higher than that of full-length protein;the intra-plate repeatability coefficient of variation is between 0.90%and 7.33%,and the inter-plate repeatability coefficient of variation is 0.84%?5.44%;the coincidence rate with the commercial ALV p27 antibody detection kit can reach 93.48%;the kit is valid for at least 5months.This study uses the recombinant protein to immunize rabbits to obtain serum antibodies with a titer of 8.1×10⁵?5.12×10⁷,the obtained antibodies can be used for Western blotting analysis(Western blotting)to identify ALV-K gp85 protein and indirect immunofluorescence analysis(IFA)to recognize the ALV-K virus in the cell.Through cell fusion and multiple subcloning screening,two hybridoma cell lines secreting antibodies were obtained,named ALV-K30B and ALV-K23H.The secreted antibody subclasses were Ig G2b and Ig G1.The prepared ascites antibody titers can be up to 2¹⁴ and 2¹²,the antibodies produced can be used for Western blotting to specifically recognize ALV-K gp85 protein and IFA to recognize ALV-K virus in cells,but not recognize ALV-A/J virus in cells.The above results indicate that the obtained ALV-K gp85 specific gene fragments can be successfully used to specifically detect chicken serum ALV-K antibody after inducing expression,and can also be successfully used to prepare high-titer polyclonal antibodies and monoclonal antibodies.The established serum antibody detection ELISA has good specificity,sensitivity,stability and coincidence rate;the prepared specific antibody can be used for the specific detection of ALV-K virus and its gp85 protein.