利用碱性磷酸酶融合表达技术研究神经导向因子netrin-4的功能

Application of alkaline phosphatase tag in functional study of axon guidance factor netrin-4

目的:采用碱性磷酸酶(AP4)标记技术研究netrin-4的神经导向功能和受体结合特征,并评估碱性磷酸酶作为标签分子在真核基因功能研究中的意义。方法:将netrin-4全长及3个结构域编码区cDNA克隆到碱性磷酸酶表达载体APtag4,成功构建真核表达载体AP4-netrin4、AP4-LNT、AP4-EGFL和AP4-NCT,进而在COS7细胞中表达出含有AP标签的融合蛋白,并通过神经组织块培养技术和神经元原代培养技术对其活性进行鉴定。结果:确认了netrin-4的神经突起导向功能。进一步通过对AP标签进行检测,发现netrin-4全长、LNT和NCT可与原代培养的神经元表面相结合,而EGFL不结合,说明神经元表面含有netrin-4受体或结合位点,且可能通过LNT和NCT结构域起作用。结论:netrin-4具有明确的神经突起导向功能,在神经元表面含有结合位点,同时发现碱性磷酸酶标签融合表达技术在真核基因功能研究方面具有一定优势。

Objective：To evaluate the performance of alkaline phosphatase（AP）-tagging technique in functional study of eukaryotie genes and study of the axon guidance activity and receptors of netrin-4. Methods ： The eDNA sequence coding netin-4 and the three domains were subcloned into the alkaline phosphatase vector APtag4 to allow successfull expression of the alkaline phosphatase tagged proteins m COS7 cells. The activity of netrin-4 was identified using cultured neuronal explants and neurons. Results：The netrin4 had axon guidance function. The AP4-netrin4 fusion protein was also found to be able to bind to the primary cultured neuron by examining the activity of AP tag. The AP4-LNT and AP4-NCT were able to bind to the primary cultured neuron, but AP4-EGFL and AP4 alone not. Conclusion： There are receptors for netrin-4 in the surface of primary cultured neuron, and the key binding domains are LNT and NCT. The strategy using AP4-tag is a powerful technique for functional study of eukaryotic genes.