小鼠耳蜗组织NT3基因的克隆及其原核表达鉴定

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，克隆人pUC19载体，行酶切鉴定和测序；然后进一步克隆人原核表达载体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 NT3 genes

AIM To clone and identify NT3 gene, very important neurotrophins, from normal cochlea of mouse for advanced 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. RESULTS We got 777 bp gene seginent by RT PCR and the DNA sequence was identical to mice NT 3 gene sequence of GenBank. This pDH expression vector was constructed successfully and NT 3 protein was expressed through temperature induction. CONCLUSION We have successfully cloned NT3 gene and constructed pDH NT3 expression vector, and also laid a basis for further study.