NT-3基因克隆与表达

Cloning and expression of human neurotrophin-3 gene

目的 :探讨利用基因工程技术制备神经营养素 3 (Neurotrophin 3 ,NT 3 )的方法。方法 :根据NT 3cDNA序列设计一对引物 ,采用聚合酶链反应 ,扩增编码人NT 3基因 ,通过基因重组 ,构建表达载体PBV2 2 0 NT 3。自动序列分析测定TN 3序列。将重组PBV2 2 0 NT 3转化到大肠杆菌DH5 α中 ,温度诱导目的基因表达。透视电镜观察诱导后的工程菌。薄层凝胶扫描分析目的基因表达量。Westernblot鉴定其免疫活性。结果 :序列分析结果与文献报道一致 ;目的基因在温度诱导下表达量为 2 5 % ,表达产物以包涵体形式存在于工程菌菌体两端 ,大小不一 ;Westernblot阳性。结论 :本实验利用基因工程技术制备NT 3是可行的 ,且表达量为 2 5 % ,表达产物具有免疫活性 ,为进一步研究其生物学功能奠定基础。

Objective: To explore the production of neurophin 3 by means of genetic engineering technique.Methods: Neurotrophin 3(NT 3) gene was amplified by PCR with primers according to its cDNA sequence.PCR products were cloned into PBV 220 . After screening and sequencing the recombined PBV 220 NT 3 were induced to express the desired NT 3,which was then determined by Western blot.The electromicroscope observation of host cell and gel scanning were also performed. Results: The NT 3 gene were successfully amplified and expressed with a yield of 25% of total protein.The expressed products existed in inclusion bodies in different size and located in the two ends of host cells.It can be recognized by anti NT 3. Conclusion: Preparation of NT 3 by genetic engineering technique is feasible and the expressed products have antigenicity.All these can provide basis for the further study of NT 3 gene's biological function.