骨肉瘤cDNA文库的构建和筛选

Construction and screening of cDNA library of osteosarcoma cells

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.