Isolation And Identification Of Avian Leukosis Virus Subgroup K And Preparation Of Monoclonal Antibodies Against Gp85 Protein

Avian leukemia(AL)is an avian tumor disease caused by Avian Leukosis Virus(ALV).It is one of the common immunosuppressive diseases in poultry.Avian leukosis viruses that occur in chickens have been divided into seven subgroups,A,B,C,D,E,J and K,of which ALV-K is identified in recent years and mainly exists in East Asia.ALV-K is reported in Chinese local chicken breed frequently.The whole genome of ALV is about 7.3kb and the structural proteins are encoded by gag,pol and env genes.The envelope surface protein gp85 determines viral subgroup specificity of the ALV.It plays an important role in ALV infection.This study includes the isolation,whole gene analysis of ALV-K strain,the expression of gp85 protein,the preparation of monoclonal antibody against gp85 protein and the epitope identification.1.Isolation,Identification and Genome Analysis of Avian Leukosis virus subgroup KThree exogenous ALV strains with weak replication ability were isolated from chicken samples which came from different regions.The viral subgroups were identified by PCR and genetic evolution map according to the gp85 gene sequence of the isolates.Our results showed that they were three ALV-K strains,named JS2110-1,hn2112-11 and AHXC2202-2.The full length of the whole gene sequence was 7493 bp,7486 bp and 7499 bp respectively.The genetic evolution map of the whole gene showed that the isolate JS2110-1 belonged to the same branch with DT190903 and JS13LY19 strains,and the homology was as high as 99.2%and 99%,respectively.The isolate hn2112-11 belonged to the same branch with GD1701 strain,and the homology was as high as 99.4%.The isolate AHXC2202-2 belonged to the same branch with DT190904 strain,and the homology was as high as 99.1%,which showed that there was a geo-genetic relationship among these strains.The length of LTR of 3 isolates was 277 bp to 285 bp,and the homology among 3 isolates was 93.8-98.9%.The length of gag gene was 2106 bp and highly conserved,compared with the reference strains,the homology was 95.1%to 99.7%.The length of pol gene was 2688 bp,the homology was 96.8%to 99.7%.The length of gp85 gene of JS2110-1 and AHXC2202-2 was 1002 bp,while the length of gp85 gene of hn2112-11 was 1005 bp,compared with the reference ALV-K strains,the homology was 93.9%to 99.9%.The length of gp37 gene were all 609bp and the homology was 94.4%to 99.7%.The successful isolation of three ALV-K strains provided biomaterials for the expression of ALV-K gp85 gene and the preparation of monoclonal antibodies against gp85 protein.2.Eukaryotic expression of ALV-K gp85 gene and preparation of its monoclonal antibodyIn order to develop monoclonal antibodies against ALV-K gp85 protein,the gp85 gene of the isolated strain AHXC2202-2 was amplified by PCR,and cloned into the pCAGGS-RIgG vector preserved in our laboratory by one-step homologous recombination to construct the recombinant plasmid pCAGGS-ALV-K gp85-RIgG with rabbit IgG Fc.The recombinant plasmid was transfected into HEK-293T cells to express the fusion protein.The recombinant expressed protein was analyzed by Western-blot with HRP conjugated Goat anti-Rabbit IgG antibody and Mouse against ALV-K gp85 polyclonal antibody.The result showed a specific band of about 120 kDa.The fusion protein was purified by HiTrap Protein G HP antibody purification column and analyzed by SDS-PAGE.Mice were immunized with the purified fusion protein.Positive hybridoma cells were screened in DF-1 cells infected with ALV-K by IFA.Four hybridoma cell lines stably secreting antibodies against ALV-K gp85 protein were successfully obtained,and named mAb ALV-K-gp85-3H6,mAb ALV-K-gp85-1E1,mAb ALV-K-gp85-1B11 and mAb ALV-K-gp85-5A4.Western blot results showed that these four mAbs had excellent reactivity and specificity.They reacted with only ALV-K.These results provided a basis for the establishment of ALV-K detection method,identification of new antigenic epitopes of gp85 protein and study of the structure and function of gp85 protein.3.Identification of epitopes of gp85 protein of ALV-KIn order to identify the new epitopes of ALV-K gp85 protein,we used the peptide scanning technique.ALV-K gp85 gene was truncated into several fragments.E.coli prokaryotic expression system was used to express the truncated gp85 protein.The reaction of truncated protein with four monoclonal antibodies was detected by Western-blot.According to the results,two new epitopes of ALV-K gp85 protein 200FCGRVP205 and 195RGPRYFCGRVP205 were identified.These epitopes were highly conserved in some ALV-K strains,while in some strains,there were a little differences in individual amino acid sites.It provided a reference for the establishment of ALV-K detection method.