摘要
目的研究骨形态发生蛋白-2(BMP-2)在骨肉瘤发生、发展中的作用,构建SAOS-2细胞cDNA文库,寻找与BMP-2相关作用蛋白。方法从骨肉瘤细胞系SAOS-2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,线形化的pGADT7-Rec和诱饵质粒pGBKT7/HA-BMP-2共转化酵母AH109菌株在文库构建的同时进行与BMP-2相互作用蛋白的筛选;或先将文库片段,线形化的pGADT7-Rec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 blotting法进一步从体外论证BMP-2相互作用蛋白。结果构建了具有基因多样性和库容量足够大的骨肉瘤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。筛选到四个与BMP-2相互作用的阳性克隆。结论此文库的多样性和库容量均符合筛选需求。可用于骨肉瘤相关基因的进一步筛选。
Objective To construct a cDNA library ofosteosarcoma SAOS-2 cells and screen the proteins interacting with the bone morphogenetic protein-2 (BMP-2) to explore the role of BMP-2 in the development of prostate cancer. Methods The total RNA was extracted from SAOS-2 cells, from which poly (A)+RNA was purified to generate the cDNA using RT-PCR with primer SMARTIII^TM and primer CDSIIIoligo (dT). The library was constructed and screened by cotransformation of the double-stranded cDNA (dscDNA) and linearized pGADT7-Rec with the bait plasmid to the yeast AH109. The proteins interacting with BMP-2 were screened by AH109 mating with the bait sWain Y187, and the interaction was confirmed using far-Western blot analysis. Results The cDNA library of human osteosarcoma SAOS-2 cells was constructed, which was characterized by high diversity and sufficient capacity. The dscDNA size range was between 250-5000 bp, with a cotransformation efficiency of4.3×10^5 and rdcombination efficiency of 1.9×10^6, and 4.3×10^5 clones were screened. The mating efficiency was 32% and 1.0×10^6 clones were screened by the bait of BMP-2, and 4 positive clones was obtained and sequenced. Conclusion The diversity and capacity of the cDNA library meet the needs for screening genes related to prostate cancer.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2007年第12期1912-1914,1917,共4页
Journal of Southern Medical University