| The infection of Reticuloendotheliosis virus can cause not only serious tumor case but also severe immune suppression in chicken groups, on the other hand much attention was given to the contamination of Reticuloendotheliosis virus in some poultry attenuated vaccine and the vertical spreation lead to many pispute between breeder chickens and commercial chickens. Besides the eraction programs, there is no effective commercialization vaccine can be used in the control of REV. Due to REV and human immunodeficiency virus have a very similar genomic structure, REV is used as a model to study the molecular mechanisms such as the quasispecies evolution of the HIV. To investigate the Reticuloendotheliosis virus infection status in large-scale breeder chicken farms of China, 8738 serum samples from different breeder farms were collected and examined using the Reticuloendotheliosis Virus Antibody ELISA Test Kit and Indirect immunofluorescence assay, and it’s quasispecies diversity was observed by comparison in different individual in a particular poultry flocks, and finally the env subunit vaccine was constructed by prokaryotic expression and eukaryotic expression respectively to observe it’s immunological effect. The details as follow:1. Investigation of the Reticuloendotheliosis virus infection in large-scale breeder chicken farms of China by serological surveyTo investigate the Reticuloendotheliosis virus infection status in large-scale breeder chicken farms of China, 563 serum samples from 4 great-grandparents breeder farms, 6557 serum samples from 8 grandparents breeder farms, 312 serum samples from parents breeder farms, totally 7432 serum samples were collected and examined using the Reticuloendotheliosis Virus Antibody Test Kit. The results showed that, REV-antibody positive rates of great-grandparents breeder flocks, grandparent breeder flocks and parents breeder flocks were 0-31%, 0-47%, 4.23%-42.50% respectively, REV-antibody positive rate has close relationship with incidence of REV infection history and tumor complaints. This study suggests that REV infection is very common in China large-scale breeder chicken farms and the prevention of REV should be given more attention, this is the largest scale serological survey for breeder chickens in China so far.2. Comparison of Reticuloendotheliosis virus quasispecies among individual chickens within an individual flockEight chickens were randomly selected from a flock raised in Heibei Province, China, because of an usual incidence of tumor growth. They were inoculated with chicken embryo fibroblasts(CEF) and DF-1 cells for the isolation and identification, respectively, of Marek’sdiseases virus(MDV), Avian leukosis virus(ALV), and Reticuloendotheliosis virus(REV). Finally, four REV strains were isolated(HB1201–HB1204) and the env gene of each was amplified and cloned. For each strain, 10 positive clones were selected for sequence analysis of the env gene to show the quasispecies diversity and the variability of the env quasispecies of the four strains was compared. The results showed that the nucleic acid homologies of the 10 env clones from the four REV strains were 99.6%–100%, 99.7%–100%, 99.7%–100%, and 99.7%–100%, respectively, and the proportions of dominant quasispecies were 20%, 30%, 60%, and 30%, respectively. The env gene homology among the dominant quasispecies of HB1201, HB1202, and HB1203 was 100%, while the env gene of the HB1204 dominant quasispecies revealed a difference in two nucleotide bases from the dominant quasispecies of the other three strains. Consequently, there was mutation at the position of the two deduced amino acids, which demonstrated that the composition of the REV quasispecies varied among infected individuals within the same period, as the dominant quasispecies continued to evolve.3. Research on REV-env-C3 d molecular adjuvant vaccineComplement is an important part of the human immune system, against a foreign substance invasion, which complement C3 d between innate and acquired immunity plays an important role as a bridge. Antibody titers of antibody C3 d fragment and the duration is one of the cleavage fragment of complement C3, C3 molecules are cleaved fragments of complement alpha chain, it cannot be protease cleavage but still maintain the characteristics of re connected with the antigen covalently, so it has extensive immune function. It can be directly with the antigen presenting with CR2 receptors on the cell, thereby reducing the activation threshold of lymphocyte, improve the immunity of the antigen, so C3 d is considered as a molecular adjuvant efficient gene vaccine. This research uses RT-PCR method to obtain the chicken C3 d gene, then 2, 3 and 4 C3 d molecules and chicken reticuloendotheliosis virus(REV) env gene 1050 bp fragment by Eco R connection, I/Xho I by enzyme digestion and connected with the expression vector PET-30 a, SDS-PAGE and Western results showed that PET-30a-env-1050, PET-env-1050-2C3 d and PET-env-1050-3C3 d the expression of PET-env-1050-4C3 d was successful, but not expression, perhaps due to the longer fragment. Nickel column affinity chromatography showed that the purified fragments of expected size, about 90% purity. The REV env gene 1050 bp respectively with 2, 3 and 4 C3 d molecular connection, via the Eco R I/Xho I by enzyme digestion and expression carrier... |
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