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  

Electronic extension and identification of new HeLa cell ESTs differentially expressed after Shigella flexneri 2a infection

作　　者：黄留玉[1] 史兆兴[1] 袁静[1] 胡福泉[2] 

机构地区：[1]军事医学科学院疾病预防控制所,北京市100071 [2]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

出　　处：《世界华人消化杂志》2007年第17期1905-1913,共9页World Chinese Journal of Digestology

摘　　要：目的:研究痢疾杆菌福氏2a入侵前和入侵后3h的人上皮细胞差异表达的新基因.方法:采用增毒的痢疾杆菌福氏2a 2457T侵袭HeLa细胞,检查HeLa细胞中细菌的数量并用RT-PCR方法从HeLa细胞中提取总RNA,制备探针.采用含有约3000个代表新基因的芯片进行芯片杂交,分析杂交结果.将所获得的新的156条EST序列为种子序列用cDNA微阵列实验,以人类EST数据库为基础库,应用siClone软件进行EST拼接、校对并尽可能延长获得大片段或全长cDNA.选取基因组未注释的编码区序列作为新基因进行RT-PCR实验验证.结果:共鉴定到≥3倍或≤0.33的差异表达的代表新基因的EST序列45条,其中25个延伸序列鉴定为与重要的肠道功能相关的已知基因,并有3个在痢疾杆菌侵袭期间高表达的存在显著差异的新EST序列,他们分别名为:NPCCKH12、ADBCSB02和HTBAMG05,这3个基因是新的人基因或假基因.结论:鉴定出了在HeLa细胞感染痢疾杆菌前后差异表达的新EST序列,为进一步研究痢疾杆菌与寄主相互作用奠定了基础.AIM： To investigate and test differential rnRNA 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）. RESULTS： Forty-five ESTs were determined as being differentially expressed, with ≥ 3-fold or ≤ 0.33-fold changes, and 25 of these were identified as known genes involved in several important intestinal functions. Interestingly, three strikingly different cDNA fragments from unknown ESTs, name NPCCKH12, ADBCSB0 and HTBAMG05, were cloned by RT-PCR, sequenced and their expression levels were confirmed by semi-quantitative RT-PCR. We confirmed that they were new human or pseudo genes CONCLUSION： Three new ESTs from HeLa cells, differentially expressed after S. flexneri 2a infection, were identified. This investigative strategy is useful for obtaining information to be applied as a basis for further study of the interactions between Shigella and epithelial cells.

关 键 词：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 

分 类 号：R346[医药卫生—基础医学]