Preparation Of Monoclonal Antibodies Against Envelope Protein Of Avian Leukosis Virus Subgroup J And Subgroup K

Avian Leukosis(AL)is a type of infectious tumor disease caused by avian retrovirus.Avian leukemia not only causes the death of diseased chickens,but also declines in production performance of layer chickens and results in immunosuppression,lower immune response ability and infection of other diseases.Since the discovery of avian leukosis virus subgroup J(ALV-J),its host range has been expanding,from the earliest infected only broiler chickens to layer chickens,extended to indigenous chickens,and even the wild ducks.At present,ALV can almost infect chicken breeds of all strains,which has resulted in huge economic losses to the poultry industry.Since 2011,some exogenous ALV strains have been isolated from several provinces in China.they have been identified that they do not belong to any subgroup of ALV,desigated as subgroup K.The emerging of subgroup K reveals the complexity of avian leukosis virus infection in Chinese flocks.Up to now,there is no commercialized vaccine or effective antiviral drugs to prevent this disease.The eradication program of ALV is the only way to control the spread of ALV.Therefore,it is particularly important to establish the ALV diagnostic method based on subgroup specific monoclonal antibody(Monoclonal Antibody,MAb),so as to achieve the rapid clinical diagnosis.ALV contains three structural genes: gag gene,pol gene and env gene.The env gene encodes envelope protein composed of gp85 and gp37 genes.gp37 gene is highly conserved among ALV subgroups,and gp85 gene of ALV were different of each other.It is also the basis for classification of ALV subgroups.In this study,primers were designed to amplify the gp85 gene of ALV-J and ALV-K respectively and then cloned into the pET-28 a and pGEX-6P-1.The recombinant plasmids were transformed into BL21 to express and purify the fusion protein.After the obtaining of purified protein,we established indirect ELISA methods for detection of ALV-J antibody and ALV-K antibody respectively.After a series of comparative tests,the best reaction conditions for indirect ELISA detection method of ALV-J antibody as follows: 14?g/ml antigen used to coat ELISA plate with pH 9.6 CB Buffer,5%skimmed milk,1:1000 of sera as detecting samples.The working concentration of HRP-labelled Goat anti mouse IgG was 1:2000 and the reaction time was 15 minthe.The best reaction conditions for indirect ELISA detection method of ALV-K antibody as follows: 6.6?g/ml antigen used to coat ELISA plate,1:2000 of sera as detecting samples.The mice were immunized with recombinant protein GST-J-gp85,and thecells were fused by hybridoma technology.The positive cells were screened,through indirect ELISA method using HIS-J-gp85 protein as coating antigen.Finally,one hybridoma cells line,named JB7,stably secreting MAb against ALV-J-gp85 protein after five cell fusion was generated.Besides mice were immunized with recombinant protein HIS-K-gp85,and ELISA plate coated with GST-K-gp85 protein.Two hybridoma cells,named KD4,KE5,stably secreting MAb against ALV-K-gp85 protein after four cell fusion was generated.Western blot showed that the MAb JB7 could specifically recognize ALV-J-gp85 protein.IFA approved that the MAb JB7 reacted with ALV-J but not with ALV-A or ALV-K.Western blot using two MAbs(KD4 and KE5)showed that two MAbs could specifically recognize ALV-K-gp85 protein.IFA approved that two MAbs reacted with ALV-K and ALV-A but not with ALV-J.The ALV-J monoclonal antibody prepared in this study lay the foundation for the preparation of ALV-J localization diagnostic kit.ALV-AK monoclonal antibodies developed here were important for the future research of ALV-K and lay the foundation for the preparation of ALV-AK diagnostic kit.