| Avian leukosis virus(ALV)is a type of retrovirus that can cause a variety of avian tumors and immunosuppressive diseases.According to the characteristics of its envelope protein,ALV can be divided into 11 subgroups of A-K,of which ALV-J is currently the most common in China.ALV-J infection mainly causes malignant hyperplasia of chicken hematopoietic cells,myeloma,hemangioma and severe immunosuppression,which seriously restricts the healthy and sustainable development of poultry industry globally.ALV-K is a novel subtype of avian leukosis virus isolated and identified in local domestic chicken in mainland China in recent years.Compared with other exogenous ALV,although the replication ability of ALV-K is weak,its co-infection with other subgroups of ALV is not uncommon.Co-infection of different ALV subgroups increases the possibility of ALV recombination and evolution.In addition,it is worth noting that the current ALV universal detection technology is not ideal for the detection of ALV-K.There is an urgent need for highly efficient and specific detection technology for ALV-K antigen and antibody.In order to develop ALV-K specific diagnostic technology and explore the pathogenic potential of the recombinant viruses between ALV-J and ALV-K subgroup,in this study,the gp85 and env genes of ALV-K were first cloned and expressed,and polyclonal antibodies against Gp85 was generated.Moreover,the ALV-K env gene and ALV-J infectious clone plasmid were used to construct the recombinant ALV virus ALV-K-env-J1,and the pathogenicity of the recombinant ALV virus was investigated in vitro and in vivo.1.Cloning and expression of gp85 gene of ALV-K and its preparation of polyclonal antibodiesTo obtain immunogens and screening antigens for preparing anti-ALV-K Gp85 monoclonal antibodies,the ALV-K gp85 gene and env gene were cloned into prokaryotic expression vector pCold I and eukaryotic expression vector pcDNA3.1(+)respectively,named pCold-K-gp85 and pcDNA3.1-K-env after sequencing verification.pCold-K-gp85 was transformed into BL21 Escherichia coli.After the induced expression by IPTG,SDS-PAGE analysis showed the recombinant Gp85 protein was mainly expressed in inclusion bodies.The purified Gp85 recombinant protein was used to immunize BALB/c mice to prepare a polyclonal antibody against ALV-K Gp85 protein.Indirect immunofluorescence(IFA)and Western blot further confirmed that the obtained polyclonal antibody against Gp85 protein could specifically recognize Env protein expressed in DF-1 cells transfected with pcDNA3.1-K-env plasmid.All these provide materials for further exploration of the range of ALV-K host,the cell receptor and the ALV-K subgroup-specific diagnostic technology.2.Rescuing and pathogenicity of ALV-K-env-Jl recombinant virusesTo investigate the potential pathogenicity of recombinant viruses between ALV-J and ALV-K subgroups,the fragment of ALV-K env gene and the linearization vector of ALV-J1 infectious clone were first amplified by PCR to construct recombinant infectious cloning plasmid.The recombinant plasmid was transfected into DF-1 cells to rescue the recombinant virus ALV-K-env-J1,which efficiently replicated and expressed ALV-K protein Env.The viral growth kinestics in DF-1 cells and pathogenicity in SPF chickens for the three viruses ALV-K-GD,ALV-K-env-J1 and ALV-Jlwere tested.The growth curve showed that the ALV-K-env-Jl has higher replication efficiency in comparison with ALV-J1 and ALV-K-GD in DF-1 cells.The chicken infection study showed that the three infected groups all demonstrated low growth performance compared with the uninfected group.Although the positive rates of chicken viremia,p27 antibody positive rate and cloaca viral shedding in the ALV-K-env-J1 infection group were lower than those in the ALV-J1 infection group,but significantly higher than those in the wild type ALV-K-GD infection group.All these indicated that the recombination between ALV-J and ALV-K can significantly enhance the replication ability and pathogenicity of ALV-K. |
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