骨肉瘤cDNA文库的构建和筛选.pdfVIP

· 1912· 南方医科大学学报(J South Med Univ) 2007；27(12) 骨肉瘤eDNA文库的构建和筛选 邓少林，权 毅，潘显明，屈 波(成都军区总医院骨科，四川 成都 610083) 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eDNA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SMARTflITM和CDSIHoligo(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-Westernblotting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eDNA文库，双链eDNA片段的长度大小范围为 250~5000 bp。共转化的效率为4．3x10 ，重组效率为 1．9x106，筛选的克隆数为4．3x105。接合法筛选时的接合效率为 32％，筛选的克隆数为1．0xl0 。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。 关键词：骨肉瘤；SAOS．2细胞；eDNA文库；酵母双杂交 中图分类号：Q78 文章标识码：A 文章编号：1673-4254(2007)12．1912．03 Construction and screening of eDNA library of osteosarcoma cells DENGShao-lin，QUANYi，PANXian·ming，Qt3Bo General Hospital ofChengdu Command，Chengdu 610083，China Abstract：Objective To construct a eDNA library ofosteosarcoma SAOS．2 cells and screen the proteins interacting with the bone morphogenetic protein-2 (BMP-2)to explore the role ofBMP-2 in the development ofprostate cancer．Methods The total RNA was extracted from SAOS-2 cells．from which poly(A)~RNA was purified to generate the eDNA using RT-PCR with primer SMARTIWu and primer CDSllIoligo(dT)．The library was constructed and screened by cotransformation ofthe double．stranded eDNA (dscDNA)and linearized PGADT7．Rec with the bait Plasmid to the yeast AH1 09．The proteins interactingwith BMP-2were sCreened byAH109matingwiththe bait strainY187，andtheinteractionwas confirmed using far-Western blot analysis． Results The eDNA 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 b