具有多种剪接形式的RNA结合蛋白基因的克隆、表达及其细胞内定位

目的：构建具有多种剪接形式的RNA结合蛋白（RBPMS）基因的真核表达载体,并在真核细胞中进行表达,确定RBPMS在细胞中的定位.方法：采用PCR技术,从人卵巢文库中扩增RBPMS基因的完整编码序列,将所扩增基因克隆到带FLAG标签的真核表达载体上,转染人胚肾细胞293T,Western印迹鉴定RBPMS的表达.然后将所扩增基因克隆到带绿色荧光标签的pEGFP-C1表达载体上,转染乳癌细胞ZR75-1,观察RBPMS在细胞中的定位情况.结果：经限制性内切酶分析和DNA序列测定鉴定构建的重组表达载体正确,Western印迹实验证明RBPMS表达成功,通过激光共聚焦显微镜观察,RBPMS分布在胞质中,在40%～50%细胞中RBPMS呈聚集状分布.结论：成功构建了RBPMS基因的真核表达载体,该基因产物分布在胞质中,在40%～50%细胞中RBPMS呈聚集状分布.

Cloning, expression and cellular localization of gene of RNA binding protein with multiple splicing (RBPMS)

Objectives:To construct the eukaryotic expression vector of RBPMS gene,express RBPMS protein in eukaryotic cells and to investigate the cellular localization of RBPMS protein.Methods:The complete coding sequence of(RBPMS) gene was amplified by PCR technique from human ovary cDNA library.The product of PCR was inserted into the pcDNA3-FLAG vector.Then human embryo kidney 293T cells were transfected with the recombinant plasmid and the(expression) of RBPMS gene was identified by Western blot. The coding sequence of RBPMS gene was also inserted into the pEGFP-C1 vector with green fluorescence tag,and then human breast cancer ZR75-1 cells were transfected with the recombinant plasmid.The green fluorescence was observed by confocal laser microscope.Results:The expression vectors of(RBPMS) gene were constructed and confirmed by restriction enzyme digestion and DNA sequence analysis.The green fluorescence could be seen in cytoplasm under confocal laser microscope,and cytoplasmic RBPMS was concentrated in granular structures in 40%-50% of transfected cells.Conclusion:The eukaryotic expression vectors of RBPMS gene are constructed successfully.RBPMS protein is located in cytoplasmic,and cytoplasmic RBPMS is concentrated in granular structures in(40%-)50% of transfected cells.