Construction And Expression Of Env-EGFP Fusion Expression Vector Of Alv-J Hb₂₀₁₁

ALV-J (Avian leukosis subgroup J) is a myeloid leukemia disease which causes several kinds of tumors in the flocks. This disease has spread all over the world,which led to huge economic loss to the poultry industry in U.S.A. and U.K,since it was discovered. It spread into the flocks in China since1999. It was reported that this disease was found in some areas in Hubei province. The pathogenicity of new strains are quite different from prototype strain HPRS-103because of the past20years’ evolution and mutation of ALV-J. Meat-type chicken was first infected with it, then the layers, and the local chicken at last. Besides, ALV-J caused myeloblastosis, histiocytosarcoma, hemangiomatosis, fibroma and erythroblastome except the myeloid leukosis. Previous studies showed that the differences of tumorigenicity of ALV is closely related to env, because the main antigen epitope and the neutralization reaction sites are located in the SU region of env. Meanwhile, the process of virus-cell fusion which cause the tumors in the organism are located in the TM region of env. In the genome of ALV-J, the gp85which encodes the SU subunit shows only40%identity to the other subgroups, however, these subgroups show80%to85%identity to each other. In addition, the gp37which encodes the TM subunit shows only65%identity to the other subgroups, but these subgroups show92%to95%identity to each other. It suggested that the env plays the leading role in causing myeloid tumor, and the Env envelope protein may play a crucial role in the tumorigenicity of the virus.In this study, the env of ALV-J HB2011strain was cloned. The forecast of amino acid sequence and the sequence homological comparison were done after obtaining the sequence. The PCR assays using the primer which contains the restriction sites of EcoRI/BamHI and BamHI/SalI sets specific to obtain the env and EGFP. These two genes were conected with the pFastBac Daul baculovirus expression vector through the restriction sites in order to construct the recombinat pFastBac-env-EGFP plasmid. Then, the plasmid was transposased into DH10Bac host bacteria in order to construct the Bacmid-env-EGFP recombinant shuttle vector. Lastly, the vector was transfected into the Sf9Spodoptera frugiperda ovary cell by liposomal transfection reagent CellfectinII in order to carry out the expression of env-EGFP fusion protein.In this experiment, a positive fragment about2200bp was obtained by the PCR assay and the env of ALV-J and the3’UTR of the HB2011strain were cloned into the E.coli. The env of HB2011strain was identified after obtaining the result of nucleotide sequencing.33strains of ALV-J from7endemic areas and the United States. Russia were selected to compare with HB2011strain(80%of them are the past four years ones). Comparing with most of the strains in China, HB2011strain showed high identity to them, especially the sdau1002strain, with98.7%identity to it. Comparing with the epidemic strains isolated from Heilongjiang, Shandong, Jiangsu, Beijing, Guangdong, Inner Mongolia and Sichuan, it showed more than93.3%identity with all of them, except the Heilongjiang JL09H01strain. Comparing with the epidemic strains isolated from the United States and Russia, it showed more than93.6%identity with all of them. It showed93.6%identity to the prototype strain HPRS-103from U.K. It is in line with worldwide trend. The analysis of amino acid sequence of the env was consist of the nucleotide analysis. Because of the base substitution and transversion in the whole env of HB2011strain, the amino acid sequence also showed variation in different degrees, which is in line with the law of the variation of strain from China and abroad. However, there were some variations that none of the strains were in line with it.The construction of pFastBac Daul-env-EGFP recombinant transfer vector and Bacmid-env-EGFP recombinant shuttle vector were confirmed by both PCR assay and enzyme digestion assay. The env-EGFP had successfully expressed in Sf9cell through the Bac-to-Bac system. Sf9cells which were transfected by the Bacmid-env-EGFP showed obvious cytopathic after3d and green fluorescence under the fluorescence microscope after8d. After passaging the virus, most of the Sf9cells which were infected with P2-generation recombinant virus showed green fluorescence under the fluorescence microscope after3d. Expression products has antigen-specific of ALV-J, since it can react with ALV-J monoclonal antibody JE9. Western-Blot result showed that the molecular weight of expression product was about90KDa.This study has important significance in finding the the receptors in different tissues and antigen-coated kit to the Env envelope protein of ALV-J.