| Within the identified genes of Marek's disease virus(MDV), 132-bpr, pp38 and meq are considered to be oncogenicity-related genes. 132-bpr and meq are only found in the MDV-1. pp38 was found to have immunosuppressive effect on chicken in vivo and the copy number of 132-bpr was found to be associated with the attenuation of the virus in vitro, meq was believed to be a potential oncogene, based on its leucine zipper structure and proline-rich domain characteristic of jun/fos family of transcription factors, and may plays an important role in the pathogenicity or oncogenicity of MDV.In the project meq gene and its protein product were studied via 3 different aspects and the main results were presented in the following:1. The comparison of meq gene sequences of different pathotypes of Marek's disease virusmeq genes of different pathotypes of Marek's disease virus (MDV) were amplified, inthe whole opening reading frame (ORF) by polymerase chain reaction (PCR) technique, sequenced and compared with the published sequence of GA strain(representing vMDV). CVI988/Rispens and 814, commercial vaccine strains popularly used worldwide and in China respectively; 648A, representing very virulent plus (vv+MDV) and 6 field isolates, originated from Guangxi commercial chickens with visceral lymphomas and vaccine breaks, representing high pathogenic (hpMDV) and two of them(G2 and N) were proved to be vvMDV, were used. The comparative analysis of sequences indicated that the sequences of meq in different pathotypes are relatively conserved and the homology of the amino acid sequences is very high. The significant differences include two mutations in both MDV-1 vaccine strains CVI988/Rispens and 814 strain: the deletion of a proline (No.l93Aa), and this mutation is just exactly located in a 15-amino-acid (EELCAQLCSTPPPPI) repeat sequence within the C-terminal transactivation domain of MEQ protein; And a pointmutation with a shift from alanine (A) of all virulent strains to serine (S) was occurred on the No.71 Aa. Two types of repeat sequence, a 15-amino-acid (EELCAQLCSTPPPPI) with 2 repeats and a 6-amino-acid (PPICTP) with 4 repeats, were firstly reported.2. The characterization of meq gene product and its expression within the cellsA recombinant baculovirus transfer vector pBlubac4-meq was constructed by cloning meq gene of Marek's Disease Virus (MDV) GA strain into the baculovirus transfer vector pBlueBac4 under the polyhedrin promoter. The recombinant meq-baculovirus was obtained by co-transfecting the insect SF9 cells with pBlubac4-meq and linearised Bac-N-Blue DNA. The recombinant baculovirus was selected by plaque assay and confirmed by PCR technique and sequencing of the inserted gene. The protein product of meq gene was highly expressed in the nuclei of recombinant baculovirus infected SF9 cells when using an anti-MEQ monoclonal antibody (McAb) 23B46 to run the immunofluorescence assay (FA); The expression quantity and IF staining patterns differed with different times post-infection (PI). The results of Western blotting and immunoprecipitation test showed there were two specific bands around 60 kD. The results of the study demonstrated that the baculovirus/insect cell system is effective to be used to express nuclear protein of virus.MEQ protein, highly expressed in the insect cell line SF9 by the baculovirus vector was immunized into BALB/c mice and the immunized spleen cells were collected and fused with the tumor cell line SP2/0 via PEG-1000 in vitro. The hybridoma cells were cloned and screened for the ability of anti-MEQ McAb secretion by FA with the MDV GA infected chicken embryo fibroblast (CEF). Four McAbs were developed and can detect the meq gene expression in the CEF infected with oncogenic MDVs and naturally occurred MD tumor cells by FA and immunohistochemistry technique respectively, but can't detect that in the CEF and normal chicken liver tissue respectively infected with non-virulent vaccine virus CVI988/Rispens.The results demonstrated that meq is highly expressed in t... |
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