人前列腺癌cDNA文库的构建和筛选  

Construction and screening of cDNA library of prostate cancer cell

作　　者：张惠[1] 庞博[1] 王健[1] 周建光[1] 李杰之[1] 黄翠芬[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850

出　　处：《军事医学科学院院刊》2005年第2期105-108,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家"863"计划资助项目(2002AA223061);国家自然科学基金资助项目(30070296)

摘　　要：目的:PC1基因是与前列腺癌相关的新基因,在雄激素非依赖和转移的前列腺癌晚期细胞系C42中高表达。为了进一步研究PC1基因的功能及作用机制,构建C42细胞的cDNA文库,寻找与前列腺癌相关基因PC1相互作用的蛋白。方法:从前列腺癌细胞系C42中提取总RNA,进而分离poly(A)+RNA,用poly(A)+RNA进行反转录并以SMARTⅢTM和CDSⅢoligo(dT)为引物进行PCR扩增,得到两端具有同源臂的PCR片段,以此同源臂为基础在酵母中实现同源重组。通过文库片段、线性化的pGADT7Rec和诱饵质粒pGBKT7PC1C共转化酵母AH109菌株,在文库构建的同时进行与PC1相互作用蛋白的筛选;或先将文库片段,线性化的pGADT7Rec转化AH109,再利用AH109和Y187两种酵母菌株的接合生殖进行筛选。最后用Far Western印迹方法进一步从体外论证了PC1蛋白可与自身相互作用形成二聚体。结果:构建了具有基因多样性和库容量足够大的人前列腺癌cDNA文库,双链cDNA片段的长度大小范围为250～5000bp。共转化的效率为4.3×105,重组效率为1.9×106,筛选的克隆数为4.3×105。接合法筛选时的接合效率为32%,筛选的克隆数为1.0×106。筛选到4个与PC1蛋白相互作用的阳性克隆。从体外证明了PC1蛋白可形成二聚体。结论:此文库的多样性和库容量均符合筛选需求。可用于前列腺癌相关基因?Objective:To construct cDNA library of prostate cancer cell line C4-2 for exploring the function and mechanism of PC-1 gene in the development of prostate cancer. Methods: Total RNA was extracted from C4-2 cells, then poly(A) + RNA was purified from the total RNA. The cDNA was synthesized from poly(A) + RNA of the prostate cancer cell line C4-2 and amplified using primer SMARTⅢ TM and primer CDSⅢoligo(dT) as the base of recombination. Then the library was constructed and screened by cotransformation of the dscDNA and linearized pGADT7-Rec with the bait to the yeast AH109. At the same time, the dscDNA and linearized pGADT7-Rec were transformed to AH109, then the interacting proteins were screened by AH109 mating with the bait strain Y187 . Far-Western blot was used to confirm the interaction. Results: The cDNA library of human prostate cancer cell line C4-2 was constructed with high multiplication and good capacity. The dscDNA size range was between 250-5 000 bp.Cotransformation efficiency was 4.3×10 5 and recombination efficiency was 1.9×10 6, and 4.3×10 5 clones were screened. Mating efficiency was 32% and 1.0×10 6 clones were screened by the bait of PC-1.Four positive clones were obtained and sequenced.It was further confirmed that PC-1 existed as dimers. Conclusion: The multiplication and capacity of the cDNA library is qualified enough to screen genes related to the prostate cancer. The proteins that have been screened require further identification and study.

关 键 词：前列腺肿瘤 C4-2细胞 基因文库 酵母双杂交 

分 类 号：R737.25[医药卫生—肿瘤]