AY887902全长扩增及其生物信息学研究

Full-length Amplification and Bioinformatics of AY887902

目的 ①对表达序列标签（EST）片段BQ135230 进行全长扩增并应用生物信息学推测其开放阅读框（ORF）、染色体定位和功能.方法 ①从新鲜的膀胱肿瘤组织中提取RNA,并用快速cDNA 末端扩增技术（RACE）方法获得BQ135230的5′端和3′端的DNA序列,然后应用GeneTool 软件合成RACE测序结果.②根据NCBI 提供的ORF Finder 程序、电子-聚合酶链反应（e-PCR）程序、Blastn、Blastp,同时应用ExPASY ProtParamTool、JUFO 软件并结合Primer 软件对AY887902全长及其相应蛋白AAX14401进行分析.Sequin 软件编辑前述结果,上报NCBI GenBank,获取基因库获得序列号（GenBank accession number）.结果 通过RACE 得到了BQ135230 的5′和3′端,GeneTool 合成一个774 bp 的全长序列.该序列在GenBank 获得序列号（accession num-ber）：AY887902,初步分析含有完整的ORF 和poly A 尾.ORF 翻译的多肽序列获得蛋白ID 号AAX14401;染色体定位11q13,与GSTP1 基因位置完全一致.AY887902 的193~195 位编码赖氨酸的碱基AAA 突变为终止子TGA,从而使有210个氨基酸的GSTP1突变为只有52个氨基酸的多肽,该假定蛋白不是跨膜蛋白和信号肽.结论 ①获得BQ135230的全长序列AY887902,推测该序列是GSTP1基因的突变体.②AAX14401在膀胱肿瘤的表达可能是膀胱癌发生发展的新的机制,为膀胱癌Ⅱ类癌提供了分子学标志,同时它有可能成为新的治疗靶点.

Objective To expedite full-length amplification of BQ135230, an EST fragment, and to postulate the open reading frame （ORF）, locating of chromosome and function by using the algorithm of bioinformatics. Meth-ods The mRNA extracted from fresh bladder cancer tissue was amplified by using rapid amplification of cDNA ends （RACE） to obtain the 5＇ and 3＇ flanking sequence of BQ135230, which entailed the computerized synthesis of the full-length sequence of AY887902 by using Genetool software. The full-length sequence of AY887902 and the protein sequence, AAX14401, were analyzed by using the ORF finder program, e-PCR program, Blastn, Blastp procedure which were provided by NCBI, as well as ExPASY-ProtParam Tool and JUFO. The results were edited by Sequin pro-gram and applied for a GenBank accession number following the report to NCBI GenBank. Results The 774 bp se-quence was synthesized by Genetool program, and the 5＇ and 3＇ flanking sequence of BQ135230 was obtained by as-sembling the result of RACE. This fragment was assigned a GenBank accession number-AY887902 and had an ORF and poly A tail. An ID code of AAX14401 for the polypeptide sequence translated from the ORF was derived （chro-mosome location： 11q13）, which corresponded to the location of GSTP1 gene. The AY887902 exhibited T-to-A and G- to-A mutation at nucleotides 193 and 195, respectively, resulting in a mutation from AAA （coding lysine） to TGA （stop codon）. No transmembrane regions or signal peptides were detected by using bioinformatic algorithms. Conclusion The full-length sequence, AY887902, is postulated to be the variant of GSTP1 gene. The AAX14401 expression pro-vide may be a novel molecular mechanism of the initiation and progression of bladder cancer and might offer a molecular marker and therapeutic target for highly malignant class Ⅱ bladder cancer.