【摘要】 目的:克隆大鼠神经营养因子4全长基因,构建真核细胞表达质粒。方法:实验于2004-06/2005-12在昆明医学院神经病学研究所完成。选用成年SD大鼠,用反转录聚合酶链反应以大鼠海马总RNA为模板,应用基因重组技术将大鼠神经营养因子4的全长基因克隆到真核表达载体pcDAN3中,构建重组表达质粒。用限制性内切酶酶切分析和DNA测序对重组质粒载体pcDNA3-神经营养因子4进行鉴定。结果:提取的总RNA电泳出现18S和28S两个条带,聚合酶链反应扩增目的基因为720bp的条带,测序结果为720bp。重组质粒的测序结果经比对与GeneBank上报道的序列一致(M86742),说明神经营养因子4基因已成功克隆至pcDNA3载体中。结论:正确的克隆了大鼠神经营养因子4基因全序列。更多还原

【Abstract】 AIM:To clone rat neurotrophin-4(NT-4)total gene and construct expression plasmid for prokaryotic cells.METHODS:The experiment was carried out in the Institute of Neuroscience,Kunming Medical College from June 2004 to December 2005.The adult SD rats were adopted in this study as models.The coding sequence of rat NT-4 total gene was amplified from the rat hippocampus total RNA by reverse transcription polymerase chain reaction(RT-PCR).By gene recombination technique,the target gene was inserted into expression vector pcDNA3 to reconstruct the expression plasmid.And the recombinant plasmid vector pcDNA3 and NT-4 was verified with restriction enzyme digestion analysis and DNA sequencing.RESULTS:Total RNA showed two bands:18S and 28S in electrochromatography,and RT-PCR product showed 720 bp band while the DNA fragment size was 720 bp.The sequencing result of recombinant plasmid was the same as the rank reported by GeneBank(M86742),which indicated that NT-4 had been cloned into pcDNA3 vector successfully.CONCLUSION:The total sequence of NT-4 has correctly cloned in rats.更多还原