树鼩肝组织cDNA文库的快速构建

为了研究动脉粥样硬化不易感动物树多种载脂蛋白的基因分子结构，我们在国内外首次快速构建了树鼯肝组织ｃＤＮＡ文库。采用一步法提取树鼯肝组织总ＲＮＡ；以寡脱氧胸苷酸纤维素为吸咐介质，柱层析法进一步纯化ｍＲＮＡ。以此为模板。利用改良的ｃＤＮＡ快速合成方法，反转录合成树鼯肝组织ｃＤＮＡ第一、二链。对ｃＤＮＡ双裢末端修饰，连接含双酶切位点的连接子后重组于噬菌体载体，包装后获得２．１ｘ１０６高滴度的树肝组织ｃＤＮＡ文库，放射自显影显示合成的ｃＤＮＡ产物大小在４００－５０００个核苷酸之间，该文库的克隆重组率为９８．５％以上，扩增随机提取的白色噬菌斑ＤＮＡ，电泳鉴定均有ｃＤＮＡ插入片段。以制备的两种多价抗血清为探针，从构建的ｃＤＮＡ文库中筛选克隆出完整的树ａｐｏＡＩ和ａｐｏＣＩ的ｃＤＮＡ核苷酸序列。与常规方法比较，本法简便、快速、重复性好，易于操作和掌握，值得推广使用。

Construction of Liver Tissues cDNA Library of Tree Shrew Lu Xinyue; Chen Baosheng; Wang Keqin ( Institute of Basic Medical Sciences, Chinese Academy of Medicd Sciences, Beijing100005) Abstract Aim To study the gene structures ofapolipoproteins of tree shrew (TS) insusceptible to atherosclerosis,the cDNA libraries of tree shrew liver tissues were quickly construeted at the first time . Methods With single-step method , total RNA was isolated from TS liver tissue, following purification of mRNA which passed through oligo-dT cellulose chromatography . Using mRNA as the template , the first and second strands of the cDNAs were Synthesized with Time Saver cDNA synthesis Kit. After been ligated with EcoRI/NotI adapters, the clDNAs were cloned into the arms of gt10or gt11 vectors. Then, therecombinantsof DNA were packed by package exbact to infect E. coli Y1090 strain . Results TS liver cDNA libraries with high titer were obtained and rate of positive recombinants was over 98.5%. Autoradiography showed that size of cDNA products were in 400-5 000 bp . Each white plaque DNA which was identified by PCR contained the cDNA insert. Full length cDNA sequence ofTS apoAI and apoCl were cloned and sequenced from the cDNA library constructed . Conclusion Compared with conventional method of constructing cDNA library the present one is simple, time-saving and easy to be used. Some problems of using the method to construct cDNA library.