LRRC4融合蛋白的构建与表达研究

Preliminary Study of LRRC4 Protein: Bioinformatic Analysis, Fusion Expression in Eukaryote and Prokaryote

在前期工作中 ,采用EST介导的定位候选克隆策略 ,克隆了一个在脑瘤中表达下调的脑特异表达新基因LRRC4 ,为进一步研究其结构与功能的关系 ,构建了含LRRC4基因全长编码区的 pGEM TEasy质粒 ,在此基础上通过亚克隆构建了LRRC4融合蛋白的绿色荧光蛋白 (pEGFP C1)表达质粒 ,瞬时转染哺乳动物细胞 ,结果发现表达的LRRC4融合蛋白定位于活细胞的细胞膜上 .同时 ,构建了LRRC4全长和截短型原核表达 pGEX 4T 2质粒 ,成功而高效地在大肠杆菌BL2 1中表达LRRC4融合蛋白 .上述工作为制备多抗 。

In previous study, a novel gene, LRRC4, a member of leucine-rich repeat (LRR) superfamily was cloned. Expression analysis indicated that LRR may play an Important rote in the central nervous system. To investigate the function and the structure-function relationship of LRRC4, full length coding region was amplified and subcloned into pGEM T Easy vector. Further, the recombinant plasmid, pEGFP-C1 /LRRC4, was constructed and transfected transiently into U251 cell. Under the fluorescence microscope, the green fluorescence produced by LRRC4 fusion protein was observed on the cytoplasmic membrane. Consistent to prediction by bioinformatics, this result indicated that product of LRRC4 is a membrane protein. In addition, the recombinant of LRRC4, pGEX-4T-2/LRRC4 and truncated LRRC4 recombinant, pGEX-4T-2/mLRRC4, were constructed and transformed into E. coli BL21. Induced by 0.5 mmol/L IPTG, The band corresponding to fusion protein were observed in SDS-PAGE as expected. Together with bioinformatic analysis of LRRC4 protein, these results establish the basis for functional study of LRRC4.