论文部分内容阅读
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?
PURPOSE: The PC1 gene is a novel gene associated with prostate cancer that is overexpressed in the androgen-independent and metastatic prostate cancer cell line C42. In order to further study the function and mechanism of PC1 gene, we constructed a cDNA library of C42 cells and searched for proteins that interact with prostate cancer related gene PC1. METHODS: Total RNA was extracted from the prostate cancer cell line C42 and further poly (A) + RNA was isolated. Reverse transcription with poly (A) + RNA and PCR amplification with SMARTⅢTM and CDSIIIoligo (dT) End PCR fragment with homology arms to achieve homologous recombination in yeast based on this homology arm. Through library fragment, linearized pGADT7Rec and bait plasmid pGBKT7PC1C were co-transformed into yeast strain AH109, and the screening of protein interacting with PC1 was carried out at the same time as library construction. Alternatively, library fragment and linearized pGADT7Rec were transformed into AH109 and AH109 and Y187 Two yeast strains were selected for mating reproduction. Finally, Far Western blotting method was used to further demonstrate in vitro that PC1 protein interacts with itself to form dimers. Results: A human prostate cancer cDNA library with gene diversity and sufficient library capacity was constructed. The length of double-stranded cDNA fragments ranged from 250 to 5000 bp. The efficiency of co-transformation was 4.3 × 105, the recombination efficiency was 1.9 × 106, and the number of clones screened was 4.3 × 105. The conjugation efficiency was 32% for conjugation screening and 1.0 × 106 for screening. Four positive clones that interacted with PC1 protein were screened. It was demonstrated from vitro that PC1 protein can form dimers. Conclusion: The diversity and library size of this library are suitable for screening. Can be used for prostate cancer related genes?