马立克病潜伏期L-meq基因对CEF细胞p53 mRNA转录水平的影响

Influence of L-meq Gene on Transcription Level of p53 mRNA in Chicken Embryo Fibroblast in Latent Period of Marek Disease

目的研究鸡马立克病(MD)潜伏期L-meq基因对鸡胚成纤维细胞(Chicken embryo fibroblast,CEF)p53mRNA转录水平的影响,并探讨L-meq基因与端粒酶活性的关系。方法构建真核转染质粒pcDNA-L-meq,转染CEF细胞,采用实时荧光相对定量RT-PCR法检测MD潜伏期L-meq基因对CEF细胞p53mRNA转录水平的影响,并用改进的TRAP法检测端粒酶活性。结果质粒pcDNA-L-meq经PCR及双酶切鉴定,均可见大小为1200bp的特异性片段。L-meq基因能激活CEF细胞中p53mRNA的转录,在转染后48和72h,p53mRNA的转录水平较相应的内参照升高,但在转染后72h,p53mRNA转录水平降低,其RQ值由48h的0.15下降为72h的0.05。转染L-meq基因48和72h后,CEF细胞中端粒酶活性均比对照组稍微升高,但在48h的端粒酶活性比72h的稍高。结论鸡马立克病潜伏期L-meq基因能微弱地刺激端粒酶活性和p53mRNA转录。

Objective To study the influence of L-meq gene on transcription level of p53 mRNA in chicken embryo fibroblast （CEF） in latent period of Marek disease and explore the relationship between L-meq gene and telomerase activity. Methods Determine the transcription level of p53 mRNA in CEF transfected with constructed recombinant plasmid peDNA-L-meq by real-time relatively quantitative RT-PCR , and the telomerase activity by a modified TRAP method. Results A specific gene fragment at length of 1 200 bp was obtained from recombinant peDNA-L-meq as proved by restriction analysis and PCR. The transcription level （RQ）of p53 mRNA in CEF 48 h after transfection（0.15） was slightly higher than that of internal reference（ GAPDH gene）, but decreased to 0.05 72 h after transfection, which was lower than that of internal reference. However, the telomerase activities in CEF cells 48 h after transfection was slightly higher than that 72 h after transfection, both of which were slightly higher than those of internal reference. Conclusion The L-meq gene in latent period of Marek disease slightly Stimulated the telomerase activity and transcription of p53 mRNA in CEF.