| 本文已被：浏览 1472次 下载 0次 |
码上扫一扫！ |
|
|
| 人尿道(阴茎)鳞癌上皮细胞(HUS-98)cDNA文库的构建及鉴定 |
|
冯爱娟1,2,杨太成1,2,刘耘1,3,冼江1,2
|
|
1. 广州军区广州总医院医学实验科,510010;华南理工大学生物科学与工程学院,510640,广州;2. 广州军区广州总医院医学实验科,510010;3. 华南理工大学生物科学与工程学院,510640,广州
|
|
| 摘要: |
| 为了进一步分离人尿道(阴茎)鳞癌组织特异性表达基因和鳞癌特异性相关基因,采用SMART技术,构建了人尿道(阴茎)鳞癌上皮细胞cDNA文库,从人尿道(阴茎)鳞癌上皮细胞中分离总RNA并纯化mRNA,利用经修饰的oligo(dT)引物合成cDNA第一链,利用SMART核苷酸作为cDNA第一链在mRNA5′端延伸出去的模板,采用LD-PCR合成双链cDNA,双链cDNA经酶切和过柱分级分离后,克隆入λTriplEx2载体后经体外包装而成cDNA文库.结果表明原始人尿道(阴茎)鳞癌上皮cDNA文库获得1.57×107个重组子,重组率达到98%.文库扩增后,滴度达到4.0×109pfu/ml,插入cDNA平均长度为2.5kb. 构建的人尿道(阴茎)鳞癌上皮cDNA文库具有良好的质量,该cDNA文库为进一步筛选鳞癌抑癌基因及鳞癌特异性表达基因奠定了基础. |
| 关键词: 尿道 鳞癌 cDNA文库 |
| DOI： |
| 分类号:Q785 |
| 基金项目: |
|
| Construction and Identification of Carcinoma Cell(HUS-98 Cell) cDNA Library of Human Urethral(Penis) Squamous Cells |
|
|
| Abstract: |
| In order to isolate and screen tissure-specific genes of human urethral(penis) squamous cell of carcinoma(SCC) and new tumor suppressor genes, the cDNA library from human urethral(penis) squamous cell of carcinoma cell was constructed by SMART(switching mechanism at 5' end of RNA transcript)technique.The total RNA and mRNA were separated from human urethral(penis) squamous cell of carcinoma cell(HUS-98 cell),and the first strand cDNA was synthesized through reverse transcription by a modified oligo(dT) primer while the SMART oligonuleotide was utilized as a template so that the first strand cDNA could be extended over the 5' end of mRNA.The double strand cDNA was amplified by LD-PCR(long-distance PCR) and then digested by Sfi I(I A & I B)restriction enzyme.After cDNA size fractionation through Chroma Spin column, the double-strand cDNA wasligated into λ TriplEx2 vector and then the recombinant DNA was packaged in vitro. The unamplified human urethral(penis) squamous cell of carcinoma cell cDNA library consisted of 1.57×10 7 independent clones in which the percentage of recombinant clones was about 98%.The titer of the amplified cDNA library was 4.0×10 9pfu/ml and the average inserts of the recombinants was 2.5kb. These results showed that the human urethral(penis) squamous cell of carcinoma cell cDNA library had an excellent quality and solid foundation for screening and cloning new tumour suppressor genes of urethral(penis) squamous cell of carcinoma and tissue-specific genes of human urethra(penis). |
| Key words: urethral(penis) squamous cell of carcinoma(SCC) cDNA library |
