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。
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).
出处
《氨基酸和生物资源》
CAS
2005年第1期56-59,69,共5页
Amino Acids & Biotic Resources
