EBV病毒全基因组cDNA探针的设计与制备

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (03): 246-250.

EBV病毒全基因组cDNA探针的设计与制备

方唯意¹, 郑文岭⁴, 马文丽³, 刘腾飞¹, 王爽², 谢卫兵², 李虹¹, 任彩萍¹, 姚开泰^1,2

1. 中南大学湘雅医学院肿瘤研究所, 湖南, 长沙, 410078;
2. 南方医科大学分子生物研究所, 广东, 广州, 510515;
3. 中南大学湘雅医学院肿瘤研究所, 湖南, 长沙, 410078;
4. 广州军区总医院肿瘤研究所, 广东, 广州 510010

出版日期:2005-03-20 发布日期:2005-03-20
基金资助:
收稿日期:2004-10-26。
基金项目:Guangzhou Science and Technology Commission as a key research project (2004z2-e0111)

Design and preparation of Epstein-Barr virus genome-wide cDNA probes

FANG Wei-yi¹, ZHENG Wen-ling⁴, MA Wen-li³, LIU Teng-fei¹, WANG Shuang², XIE Wei-bing², LI Hong¹, REN Cai-peng¹, YAO Kai-tai^1,2

1. 中南大学湘雅医学院肿瘤研究所, 湖南, 长沙, 410078;
2. 南方医科大学分子生物研究所, 广东, 广州, 510515;
3. 中南大学湘雅医学院肿瘤研究所, 湖南, 长沙, 410078;
4. 广州军区总医院肿瘤研究所, 广东, 广州 510010

Online:2005-03-20 Published:2005-03-20

摘要/Abstract

摘要： 目的设计和克隆所有EB病毒(EBV)已知和预计的编码基因作为cDNA探针用于构建EBV微阵列,以促进研究EBV在其相关疾病中的发病学作用。方法 cDNA探针首先由Oligo6.0,Blast和Primer Primier软件设计和筛选全EBV基因组探针,它们的长度被定在300~600 mer并且具有高度特异性。从B95-8细胞和NPC组织的基因组DNA和RNA中,通过PCR和RT-PCR方法扩增这些探针,之后克隆入T/A克隆载体。所有的探针被测序鉴定。结果除LF1和LF3基因在B95-8细胞的EBV基因组中不存在外,其余85个基因片段(BWRF1基因有7个重复阅读框架)和两个EBERs基因被成功克隆。结论 EBVcDNA探针的设计和克隆为制备EBV微阵列和进一步研究EBV基因组在其相关疾病中的作用奠定了基础。

Abstract: Objective To design and clone all known and predicted coding genes of Epstein-Barr virus (EBV) as the cDNA probes for preparing the microarray for EBV detection, thereby to facilitate further investigation of the pathogenetic role of EBV.Methods Oligo 6.0 software, BLAST program and Primer Premier 5 software were employed to design and screen the cDNA probes of the whole EBV genome, whose length ranged from 300 to 600 mer each with high specificity. These cDNA probes obtained through PCR and reverse transcriptase (RT)-PCR amplification from the genomic DNA and RNA of B95-8 cells and nasopharyngeal carcinoma (NPC) tissue were cloned into T/A clone vector, followed by identification of these probes by sequencing analysis.Result and Conclusion A total of 85 gene fragments (BWRF1 genecontained 7 repeats of open reading frames) coding for proteins and 2 EBERs in EBV genome were successfully cloned, not including LF1 and LF3 genes that did not exist in EBV genome of B95-8 cells, which provides the basis for preparing microarray to explore the role of EBV genome in its related diseases.

中图分类号:

R373

方唯意¹, 郑文岭⁴, 马文丽³, 刘腾飞¹, 王爽², 谢卫兵², 李虹¹, 任彩萍¹, 姚开泰^1,2. EBV病毒全基因组cDNA探针的设计与制备[J]. 南方医科大学学报, 2005, 25(03): 246-250.

FANG Wei-yi¹, ZHENG Wen-ling⁴, MA Wen-li³, LIU Teng-fei¹, WANG Shuang², XIE Wei-bing², LI Hong¹, REN Cai-peng¹, YAO Kai-tai^1,2. Design and preparation of Epstein-Barr virus genome-wide cDNA probes[J]. Journal of Southern Medical University, 2005, 25(03): 246-250.

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