关键词: NT3;克隆，分子;耳蜗;小鼠

    摘 要:目的 克隆小鼠耳蜗组织中重要神经营养因子NT3的基因，并对其进行原核表达鉴定，为进一步研究该基因对内耳听觉功能的影响以及对耳聋的基因治疗提供基础. 方法 根据所设计的引物用RT-PCR方法扩增目的基因，克隆入pUC19载体，行酶切鉴定和测序;然后进一步克隆入原核表达载体pDH，温度诱导表达，SDS-PAGE检测表达产物. 结果 RT-PCR得到长度为777bp的基因片段，克隆后酶切鉴定正确，测序结果经BLAST对比，与GenBank中小鼠NT-3序列完全一致.成功构建了原核表达载体，经温度诱导表达出NT3蛋白. 结论 从耳蜗组织中能获得正确的NT3基因克隆以及稳定的原核表达，为后续实验提供了保证.

    Cloning and identification of normal mouse cochlear NT3genes

    GAO Xia，JIN Ming，GAO Hui，JIA Lin-Tao，WANG Feng，WANG Jin-Ling，YANG An-Gang

    1 Department of Otolaryngology，Xijing Hospital，2 Department of Biochemistry&Molecular Biology，Faculty of Preclinical Medicine，Fourth Military Medical University，Xi'an710033，China

    Keywords:NT3;cloning，molecular;cochlea;mice

    Abstract:AIM To clone and identify NT3gene，very im-portant neurotrophins，from normal cochlea of mouse for ad-vanced study and treat ment or prevention of hearing loss.METHODS According to designed primers，we used RT-PCR to amplify target genes and cloned them to the vector of pUC19，then analyzed the DNA sequence of genes by DNA analysis stations and compaired them with GenBank.RE┐SULTS We got777bp gene seginent by RT-PCR and the DNA sequence was identical to mice NT-3gene sequence of GenBank.This pDH expression vector was constructed suc-cessfully and NT-3protein was expressed through tempera-ture induction.CONCLUSION We have successfully cloned NT3gene and constructed pDH-NT3expression vector ......