不易感动脉粥样硬化动物北京鸭肝cDNA文库的快速构建
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国家攀登计划资助


Construction of Liver Cells cDNA Libraries of Beijing Duck Insusceptible to Atherosclerosis
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    摘要:

    为了研究不易感动脉粥样硬化动物北京鸭多种载脂蛋白的基因分子结构,在国内外首次快速构建了鸭肝细胞CDNA文库。采用一步法提取鸭肝细胞总RNA;寡核胸苷酸为吸附介质,柱层析法进一步提取mRNA。以此为模板,利用改良的中CDNA合成方法,反转录合成鸭肝细胞CDNA的第一、二链。经CDNA双链末端修饰,连接含双酶切位点的连接子后重组于噬菌体载体,包装后获得高滴度的鸭肝细胞。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98.5%以上,随机提取白色噬菌斑DNA,酶切鉴定均有CDNA插入片段。以制备的兔抗鸭aPoAI多抗为探针,从构建的cDNA文库中克隆出鸭aPoAIcDNA核苷酸序列,与常规方法比较,本法简便、快速,易于操作和掌握。本研究还对应用此方法构建CDNA文库的某些环节进行了讨论。

    Abstract:

    Aim To study the gene structures of apolipoproteins of Beijing duck insusceptible to atherosclerosis,the cDNA libraries of duck liver cells were quickly constructed at the first time.Methods With single-step method. total RNA was isolated frorn duck liver tissue. following purification of mRNA which Passed through oligo dT cellulose chromatography. Using mRNA as the template . the first and sceond strands of the cDNAs were synthesized in Time Saver cDNA synthesis Kit. After been ligated with EcoRl /Notl adaptors, the cDNAs were cloned into the arms of λgt10 or λgt11 vectors. Then the recombinants of ADNA were packed to infect E. coli Y 1090 strain. Results Duck liver cDNA libraries with high titer were obtained and rate of positive recombinants was over 98. 5 %. Autoradiography showed that sizes of cDNA products were in 400~ 5000 bp. Each clear plaque DNA which was identified by endorestriction enzyme digestion contained the cDNA insert. Full length cDNA sequence of duck apoAI was 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 eady to be used. Some problems of using the method to construct cDNA library were discussed in detail.

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吕新跃,陈保生,王克勤,薛红.不易感动脉粥样硬化动物北京鸭肝cDNA文库的快速构建[J].中国动脉硬化杂志,1997,5(4):304~308.

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  • 收稿日期:1997-09-09
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