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目的了解SARS-COV不同毒株5’UTR RNA的构成特点和差异及其空间结构;研究该序列在真核细胞中的启动子活性。方法用软件DNASTAR-MEGALIGN和BLAST分析SARS-COV5’UTR序列同源性、RNADRAW3.0预测二级结构;构建5’-UTR CDNA序列驱动的荧光素酶基因表达质粒PGL3-5’-UTR,转染HEPG2细胞,检测萤火虫荧光素酶的表达;构建一套缺失突变质粒,使其分别3’端残留三个、两个、一个和零个(完全缺失)茎环,检测报告基因的表达;采用5’-RACE,查找转录起始位点;将PGL3-5’UTR转染A549、HEPG2、VERO E6、HELA和ECV304,检测报告基因的表达差异。结果 SARS-COV 5’UTR全长为264NT,18株有缺失突变均位于5’端。101个SAJRS-COV 5’UTR序列中,共发现5个点突变位置;SARS-COV 5’UTRRNA可形成稳定的二级结构。STEM-LOOP-Ⅱ含多个茎环结构,形成复杂的假结;PGL3-5’-UTR有明显的萤火虫荧光素酶表达;当4个茎环都具备时,荧光索酶相对活性是SV40启动子的约43%;失去第一个茎环后,是SV40启动子的约45%;而失去前两个后,则不表达荧光素酶;转录起始位点在SARS-COV 5’UTR的第56位核苷酸;在五种不同细胞中,表达强度由高到低依次是:A549、HEPG2、ECV304、HELA和VERO E6。结论①SARS-COV 5’UTR序列保守,二级结构可形成4个茎环结构域;②SARSC-OV 5’UTR有启动子活性;③在二级结构中,与启动子活性有关的结构域在前两个茎环;④SARS-COV 5’UTR在调控基因转录时,第56位核苷酸及其下游的TRS具有重要作用;⑤多种组织或器官都可为SARS-COV 5’UTR发挥启动子功能提供辅助因子,而以肺源性细胞最为适合。
OBJECTIVE: To investigate the characteristics, differences and spatial structure of 5 ’UTR RNA of different strains of SARS-COV and investigate the promoter activity of this sequence in eukaryotic cells. Methods The DNA sequence homology of SARS-COV 5’UTR was analyzed by software of DNASTAR-MEGALIGN and BLAST. RNADRAW3.0 was used to predict the secondary structure. The luciferase gene expression plasmid PGL3-5’-UTR HEPG2 cells were harvested and the expression of firefly luciferase was detected. A set of deletional plasmids were constructed so that the three, two, one and zero (completely deleted) 5’-RACE to find the transcription start site. The PGL3-5’UTR was transfected into A549, HEPG2, VERO E6, HELA and ECV304 to detect the expression difference of the reporter gene. Results The total length of 5’UTR of SARS-COV was 264NT, and the 18 deletion mutants were all located at the 5 ’end. A total of 5 point mutations were found in 101 SAJRS-COV 5 ’UTR sequences; the SARS-COV 5’ UTRRRNA could form a stable secondary structure. STEM-LOOP-Ⅱ contained multiple stem-loop structures, forming a complex pseudoknot; PGL3-5’-UTR had significant firefly luciferase expression; and when all four stem-loops were present, the relative activity of the fluorescent enzyme was SV40 About 43% of the daughter; about 45% of the SV40 promoter is lost after losing the first stem; while after losing the first two, no luciferase is expressed; the transcriptional start site is at the 5 ’UTR of SARS-COV 56th nucleotide; in five different cells, the expression intensity descending order was: A549, HEPG2, ECV304, HELA and VERO E6. CONCLUSIONS: ①SARS-COV 5’UTR sequence is conserved, secondary structure can form four stem-loop domains; ②SARSC-OV 5’UTR has promoter activity; ③ In the secondary structure, the promoter activity related domains in the former Two stem loops; ④SARS-COV 5’UTR plays an important role in regulating gene transcription when the 56th nucleotide and its downstream TRS play an important role; ⑤SARS-COV 5’UTR can play a promoter in many tissues or organs Function to provide cofactors, and most suitable for pulmonary-derived cells.