马立克氏病病毒meq基因功能研究

Function Analysis of Marek's Disease Virus meq Gene

关键词:  马立克氏病病毒  致瘤基因/meq基因  转录激活因子  序列比较  杆状病毒/昆虫细胞表达系统  RCAS载体  单克隆抗体  功能分析

Key words:  Marek's disease virus  oncogene/meq  transactivator  comparison of sequence  baculovirus/insect cell system  RCAS vector  monoclonal antibody  function analysis

摘要:

从马立克氏病病毒(MDV)不同致病型毒株meq基因序列、meq基因产物及其细胞内表达特性和meq蛋白生物学功能的研究探讨马立克氏病病毒致瘤基因meq功能。完成了648A、CVI988/Rispens、814、广西地方毒株G2、N、0093、0095、0297、0304共9个MDV毒株meq基因的序列测定。MDV不同致病型毒株的。meq基因序列相对比较保守,它们相互间核苷酸和氨基酸序列的同源性均很高;与所有7个致瘤的MDV毒株相比,在2个MDV-1弱毒疫苗CVl988/Rispens株和814株发现有二个特征性位点突变;此外,还在其ORF中首次发现含15个氨基酸残基(EELCAQLCSTPPPPI)的2个重复和含6个氨基酸残基(PPICTP)的4个重复,全分布在MEQ蛋白C-端的转录激活域内。MEQ蛋白的表达仅局限于感染细胞的核内,而且随感染时间增加,具有从核质向核仁和核膜转移趋向;Western Blotting和免疫沉淀试验证实重组杆状病毒感染细胞裂解物中有大小约为60kD的特异带。利用表达的MEQ蛋白产物免疫BALB/c小鼠,获得的杂交瘤细胞被克隆并与MDV感染的鸡胚成纤维细胞(CEF)做免疫荧光试验(FA),获得4株稳定产生抗MEQ蛋白单克隆抗体(McAb)的杂交瘤细胞,其中3G12E6单克隆抗体能够检测到MDV致瘤株感染的CEF及自然MD肿瘤细胞中表达的meq基因产物,而CVl988/Rispens感染的细胞则未检测到。发现细胞内表达的meq基因产物可明显促进MDVGA株对体外培养细胞的感染及增殖。研究结果表明,meq基因在感染细胞内的表达水平是MDV增殖和致病、致瘤的分子基础。

Abstract:

The function of meq gene of Marek's Disease Virus (MDV) was characterization of analysed by the DNA sequences comparison of different strains of MDV, characterization of gene product and its expressions within the cells and biological function of MEQ. Genes of viruses 648A, CVI988/Rispens,814 and local strains such as G2,N,0093,0095,0297,0304 were amplified in the whole opening reading frame (ORF) by polymerase chain reaction (PCR) technique. Two site mutations were found in CVI988/Rispens and 814, as two interesting types of repeat sequence, a 15-amino-acid (EELCAQLCSTPPPPI) with 2 repeats and a 6-amino-acid (PPICTP) with 4 repeats located in the C-terminal of the gene, were found in all the MDVs. The insect SF9 cells infected with the recombinant baculovirus and the cell extractions were detected by the immunofluorescence (IF),Western blotting and the immunoprecipitation,respectively. It was found that meq gene was highly expressed in the nuclei of the SF9 cells and the expression quantity and the IF staining patterns differed in different time of post-infection (PI). The Western blotting and immunoprecipitation test showed there were specific bands around 60 kD. The MEQ protein extracted from the infected SF9 cell 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 immunofluorescence assay (FA) with the MDV GA infected chicken embryo fibroblast (CEF). Four McAbs were developed and detected to reveal the expression of meq gene in the CEF infected with oncogenic MDVs and naturally occurred MD tumor cells by FA and immunohistochemistry technique respectively, but not to reveal that of the CEF and the cells of visceral organ infected with CVI988/Rispens. The expression of meq gene within the cells could enhance the infection and replication of GA and then resulted in the increase of virus plaques. The results showed that the findings lead to form a hypothesis that the expression activity of the meq gene might be the molecular basis for the replication and the subsequent pathogenicity and oncogenicity of MDV.