早老性痴呆大脑cDNA文库构建及目的基因克隆

 取临床确诊为早老性痴呆 (Alzheimer’sdisease ,AD)患者的大脑组织 ,应用磁珠法直接提取mRNA ,电泳检测其质量 .经逆转录合成双链cDNA后 ,用碱性凝胶电泳检测其大小在 0 .2～ 9.0kb范围 ,主要集中在 1.0～ 2 .0kb之间 .层析除去多余的adaptors ,收集大于 4 0 0bp的cDNA片段 ,与载体pYESTrp2连接 ,经电转化后 ,得到克隆总数为 5.1× 10 5的AD病人大脑cDNA文库 .用PCR技术从该文库中扩增得到小肠三叶因子 (intestinaltrefoilfactor ,ITF) [1] 和神经生长抑制因子 (growthin hibitoryfactor,GIF) [2 ] 的cDNA编码区 .研究表明 ,所构建的cDNA文库质量较高 ,可广泛用于AD病研究工作 .同时 ,将所克隆的GIF编码区插入到载体pHybLex Zeo上 ,构建成带饵基因的质粒 ,为进一步通过酵母双杂交方法搜寻与GIF相互作用的神经因子提供了必要条件

Construction of the Cerebrum cDNA Library of an Alzheimer's Disease Patient and Cloning of Interesting Genes

The construction and evaluation of the cerebrum cDNA library of an Alzheimer's disease(AD) patient are described. mRNA was extracted directly from the cerebrum of AD patient by paramagnetic particle method. After mRNA was reverse transcribed into double stranded cDNA, the fragments were showed in the range of 0.2～9.0 kb on the alkaline agarose gel electrophoresis. After separation on the Sephacryl S 400 spun column to eliminate excess adaptors and small fragments (less than 400 bp), the cDNA was ligated into the pYESTrp2 plasmid. Complexity of the plasmid library was confirmed to be about 5.1×10 5.It was demonstrated that the library is of high quality and can be used as a valuable source on AD research. Meanwhile the target cDNA genes such as intestinal trefoil factor (ITF) and growth inhibitory factor(GIF) were probed with PCR amplification. The cDNA gene of GIF was inserted into the pHybLex/Zeo plasmid.The results provided good material for searching interaction factors with GIF bait gene by yeast two hybrid system consequently.