Proteomic Analysis Of Bursa Of Fabricius Of SPF Chickens Infected With Marek's Disease Virus

Marek's disease virus (MDV), as an oncogenic herpesvirus, induces internal and cutaneous tumor and infects nervous system that leads to paralysis or death in susceptible chickens. Currently, Marek's Disease is kind neoplastic disease that can be successfully prevented by vaccine, which plays an important role of comparative medicine. Determining the effects of MDV infection on the chicken proteome may provide fundamental insights for tumorigenesis and reveal new and novel targets for therapeutic investigation. The bursa of Fabricius, as an important lymphoid organ, performs both primary and secondary immune functions, and it is also the site where the replication finishes. The cytolytic phase is an important phase of MDV replication cycle that mainly occurs in bursa of Fabricius. And this phase usually causes immune suppression and thus increases susceptibility to other secondary infections. This study successfully established the two-dimensional gel (2-DE) technology of bursa tissue. Then, the bursae of Fabricius from RB1B-infected chickens and control chickens at different stages were analyzed by 2-DE technology, and the levels of virus-infected were detected by RT-PCR, respectively. Some of differential spots were identified with mass spectrometry and reversion to verification. The difference proteins were analyzed by bioinformatics to discuss the significance of the difference proteins and possible mechanism of virus infected.1. Development of two-dimensional gel electrophoresis for proteomic of bursa of Fabricius.To develop two-dimensional electrophoresis (2-DE) with good repetition and high resolution for the proteomic analysis of bursa of Fabricius, in this study, the bursa of Fabricius from specific pathogen free (SPF) chickens were analyzed by 2-DE. The first dimension was isoelectric focusing in immobilized pH gradients (IPG) strip and the second dimension was SDS-PAGE on vertical electrophoresis units. The conditions were optimized, such as sample preparation, rehydration, isoelectric focusing, equilibration, staining, and other steps. The results were analyzed with the software PDQuest 8.0.1. On the 17 cm IPG strip (pH 5–8), more than 800 spots were detected in normal bursa of Fabricius tissue through this method, and the match rate of protein spots was 83.5 %. The development of the 2-DE method for proteomic of bursa of Fabricius should provide a basis for further elucidation of the development and immune function of bursa of Fabricius.2. Viral infection level in bursa of Fabricius of chickens infected with MDVMDV infection in vivo is divided into four main phases, that is, early cytolytic infection, latent infection, cytolytic re-infection, and tumor cell proliferation. The animal experiments were designed according to these stages. SPF chickens were infected with RB1B strain via intraperitoneal injection. Lesions of major immune organs were detected at different days post infection (4 DPI, 7 DPI, 14 DPI, and 21 DPI). The frozen section of the bursa of Fabricius was prepared and stained by H.E. And the relative mRNA level of MDV-Meq and MDV-gB in the bursa of Fabricius at different stages was detected by semi-quantitative RT-PCR. The results showed that the mRNA level of MDV-Meq gene had an increasing trend in 7DPI and 21DPI, especially significantly increased in21DPI; the MDV-gB gene, significantly increased in 4 DPI and 21 DPI, while the level decreased in latency period (7 DPI and 14 DPI). These data should contribute to proteomics research about host response in bursa of chickens infected with MDV.3. Proteomics of host responses in the bursa of Fabricius of chickens infected with MDVThe bursae of Fabricius of chickens infected with RB1B were analyzed by 2-DE technology, and the 2-DE maps at different stages (4 DPI, 7 DPI, 14 DPI and 21 DPI) were analyzed with software PDQuest 8.0.1. Some of differential spots were identified with mass spectrometry. The results showed 800-900 protein spots were detected in every 2-DE gels. Comparative analysis of these 2-DE gels at different teams in multiple 2-DE gels revealed that 77 differentially expression protein spots during different period. Then these spots were further analyzed by SPSS software, and twenty-nine significant and regularity change spots were selected, including 13 persistent up-regulated protein spots,10 persistent down-regulated protein spots,2 up-regulated at antephase and down-regulated at anaphase protein spots, 4 down-regulated at antephase and up-regulated at anaphase protein spots. The 29 protein spot were analyzed by matrixassociated laser dissociation / ionization time of flightmass spectrometry (MALDI-TOF-MS). Twenty-four proteins were successfully identificated, including Apolipoprotein A1, leukemia-associated protein, tumor protein 18, Ras-related protein Rab11A, HSP27, BUB3 homolog, thioredoxin domain containing 5, chaperonin containing TCP1, beta2 microglobulin, similar to DnaJ protein SB73 (HSP40), phosphomannomutase 2, heterogeneous nuclear ribonucleoprotein D-like, slow muscle troponin T, similar to LOC129607 protein, 58kDa glucose regulated protein precursor, trypsinogen, hypothetical protein(Mismatch repair ATPase,MutS family), hypothetical protein (GATase1-like domain). In addition, we discoveried two differential proteins different from those published in the database of fowl, which further study is needed. As confirmed by database query, these differential proteins were mainly associated with tumor, protein folding, signal transduction, immunology, as well as cell proliferation and apoptosis. Based on the above research, we analyzed the function of these differential proteins as well as their relationship with MDV infection mechanism and tumor through bioinformatics analysis combined with the present reports. ApoAI was found that sustained over-expression may be associated with MDV infection mechanism of virus particles on the membrane, while expression of ApoAI had led disordered lipid metabolism may be easy to eventually lead to atherosclerosis of MDV infection. LAP18 and Op18 were significantly fluctuating expression in this experiment, suggesting that they may be related to MDV-induced tumor formation of the early. The HSP27 was found that over expression in this study. The function of over-expressed HSP27 was confirmed to delay apoptosis. RB1B could induced HSP27 over-expression in order to benefit a large number of their offspring. Rab11A protein was founded that sustained up-regulated expression in this study. Rab11A protein may be associated with MDV cell transfer or copy mechanism. Rab11A was also expected to be the role of molecular target therapy to anti-herpes simplex virus. Down-regulated expression of the BMG might be related with the MDV-induced immunosuppressant in this experiment. BUB3 protein and Trx protein are related with tumor formation, which continued up-regulated expression in the four-stage. The latter difference was extremely significant that prognosticated the period of tumorous formation was coming. This study firstly reported host responses of promotes in the bursa of Fabricius of chicken infected with MDV. It was important to further study of MDV pathogenesis and viral-induced tumors provided much data, as well as made some basis messages for the MDV infection chicken as tumor model.4. The detection of mRNA of ApoAI and Op18, and Western blot validations of ApoAI in the bursa of Fabicius of chicken infected with MDVIn the preliminary study, Apolipoprotein A1 (ApoAI) was found that sustained over-expression, and Oncoprotein 18 (Op18) was significantly fluctuating expression at different stage, as indicated by 2-DE and mass spectrometry. In this the experiment, after infection with RB1B, the mRNA level of ApoAI and Op18 was detected in bursal tissue of the experimental group and control group at different time points through semi-quantitative RT-PCR. And the detected result of mRNA level was compared with that detected by 2-DE and mass spectrometry to determine their consistency. The ORF of ApoAI and Op18 were cloned into pGEX6p1 vector, respectively. Two GST-fusion proteins were obtained by prokaryotic expression. The fusion protein GST-ApoAI was recovered through electroelution, and Balb/c mice were immunized to prepare antiserums for authentication the results of 2-DE and mass spectrometry at protein level by Western blot. The results showed that the mRNA levels of ApoAI and Op18 were consistent with the results of the 2-DE and mass spectrometry analyzed at different stages. The multi-antiserums were able to effectively identify ApoAI protein in bursal tissue samples of experimental and control groups at different stages by Western blot, and the results were consistent with that of 2-DE.