人乳腺癌细胞cDNA噬菌体表达文库构建及鉴定

Construction and identification of cDNA library of human breast cancer cells

目的:进一步分离人乳腺癌组织特异性表达基因和腺癌特异性相关基因。方法:采用SMART技术,构建人乳腺癌细胞cDNA噬菌体表达文库,从人乳腺癌细胞中分离总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:原始人乳腺癌细胞cDNA噬菌体表达文库获得1.68×107个重组子,重组率达到98%。文库扩增后,滴度达到4.2×109pfu/mL,插入cDNA平均长度为2800bp。结论:构建的人乳腺癌细胞cDNA噬菌体表达文库具有良好的质量,该cDNA文库为进一步筛选乳腺癌抑癌基因及乳腺癌特异性表达基因奠定了基础。

To further isolate tissue-specific genes and new tumor suppressor genes of human breast cancer, Methods A cDNA library of human breast cancer cells was constructed by SMART （switching mechanism at 5＇ end of RNA transcript） technique, The total RNA and mRNA were separated from human urethral squamous cells of carcinoma cell （HUS-98 cell）, and the first-stranded cDNA was synthesized through reverse transcription with a modified oligo （dT） primer while the SMART oligonuleotide was utilized as a template so that the first-stranded cDNA could be extended over the 5＇ end of mRNA, The double-stranded cDNA was amplified by LD-PCR （long-distance PCR） and then digested by sfi I （ I A ＆ I B） restriction enzyme. After cDNA was fractionated through Chroma Spin column, the double-stranded cDNA was ]igated into the λTriplEx2 vector and then the recombinant DNA was packaged in vitro. Results The unamplified human breast cancer eDNA library consisted of 1.68 ~ lO7 independent clones in which the pereentage of recombinant clones was about 98%. The titer of the amplified cDNA library was 4.2 × lO9 pfu/ml, and the average insert of the recombinants is 2 800 bp in length. Conclusion These results show that the human breast cancer cDNA library has an excellent quality and lays solid foundation for screening and eloning tissue-specifie genes and new turnout suppressor genes of human breast cancer.