HeLa细胞感染痢疾杆菌前后差异表达的新EST序列的电子延伸及验证
痢疾杆菌福氏2a;表达序列标签;cDNA微阵列;电子延伸;反转录聚合酶链反应;基因鉴定,黄留玉,史兆兴,袁静,胡福泉,黄留玉,通讯作者:,ElectronicextensionandidentificationofnewHeL
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黄留玉, 史兆兴, 袁静, 军事医学科学院疾病预防控制所 北京市 100071
胡福泉, 中国人民解放军第三军医大学基础医学部微生物学教研室 重庆市微生物工程实验室 重庆市 400038
黄留玉, 研究员, 博士生导师, 主要从事微生物功能基因组学研究.
通讯作者: 胡福泉, 400038, 中国人民解放军第三军医大学基础医学部微生物学教研室, 重庆市微生物工程实验室. hooququan@yahoo.com.cn
电话: 023-68752240
收稿日期: 2007-03-26 接受日期: 2007-04-21
Electronic extension and identification of new HeLa cell ESTs differentially expressed afterShigella flexneri 2a infection
Liu-Yu Huang, Zhao-Xing Shi, Jing Yuan, Fu-Quan Hu
Liu-Yu Huang, Zhao-Xing Shi, Jing Yuan, Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing 100071, China
Fu-Quan Hu, Department of Microbiology, College of Medicine, the Third Military Medical University of Chinese PLA, Chongqing 400038, China
Correspondence to: Fu-Quan Hu, Department of Microbiology, College of Medicine, the Third Military Medical University of Chinese PLA, Chongqing 400038, China. hooququan@yahoo.com.cn
Received: 2007-03-26 Accepted: 2007-04-21
Abstract
AIM: To investigate and test differential mRNA expression of new ESTs within HeLa epithelial cells following infection with Shigella flexneri 2457T.
METHODS: HeLa cells were incubated with S. flexneri 2a 2457T. A methylene blue assay was performed to examine the ratio of bacterial infection. Total RNA was extracted from HeLa cells and mRNA was isolated for use as probes. A cDNA microarray was assembled with about 3000 cDNA clones representing the same number of independent cDNA clusters, which were unknown-gene ESTs. Using 156 EST sequences obtained from cDNA microarray analysis as seed sequences, the Siclone software was applied for splicing, proofing, and extending EST sequences as long as possible. To validate the correctness of sequences after extension and to confirm the accuracy of the differential expression of genes from the microarray analysis, three new genes were selected and their transcription levels in HeLa cells were analyzed before and after Shigella infection using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) ......
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