Cloning of Neuron Specific Protein, MxR

通过筛选小鼠脑cDNA文库，克隆了一个编码小鼠神经内分泌特异蛋白/网状蛋白（RTN）3的cDNA。mRTN 3/mMxR 1的cDNA全长为1745 nt，含有711 nt的开放阅读框（ORF）。它有832 nls长的3'非编码区（NCR），多聚腺苷酸信号（AATAAA）位于1694。序列分析表明mRTN 3/mMxR 1与人RTN在C端有很强的同源性。北方印迹分析表明mRTN 3/mMxR 1在脑内表达。其表达不受胚胎发育的控制。我们在COS 7或HeLa细胞中表达了ORF与绿色荧光蛋白的融合蛋白。表达的融合蛋白定位于内质网。当去除独特的N-末端序列时，截短的融合蛋白保留在ER中，尽管蛋白质的强度降低。我们现在试图确定mRTN 3/mMxR 1到ER膜的拓扑结构。我们还试图通过用合成肽免疫兔子来制备特异性抗体。

We cloned a cDNA encoding mouse neuroendocrine specific protein/reticulon (RTN) 3 by screening mice brain cDNA library. The cDNA of mRTN3/mMxR1 was 1745nts and contains 711nts or open reading frame (ORF). It has 832nls of long 3' noncoding region (NCR), and poly A signal (AATAAA) is located at l694. The sequence analysis indicated that mRTN3/mMxR1 has strong homology with human RTN in its C-terminus. Northern blot analysis indicated that mRTN3/mMxR1 was expressed in brain. Its expression was not controlled embryonic-developmentally. We expressed the ORF as a fusion protein with green fluorescent protein in COS 7 or HeLa cells. The expressed fusion protein was localized in the endoplasmic reticulum. When the unique N-terminal sequence was removed, the truncated fusion protein remained in the ER though the intensity of the protein was reduced. We are now trying to determine the topology of mRTN3/mMxR1 to the ER membrane. We are also trying to make a specific antibodies by immunizing rabbits with synthetic peptides.