RBPMS不同剪接体的真核表达和亚细胞定位

Eukaryotic Expression and Subcellular Localization of Different RNA Binding Protein with Multiple Splicing Isoforms

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，并在真核细胞中表达，确定不同形式的RBPMS在细胞中的定位。方法：采用PCR技术从人卵巢cDNA文库中扩增RBPMS基因的几种完整编码序列（命名为RBPl～RBP4），克隆到带绿色荧光蛋白标签的pEGFP-C1表达载体上，转染人胚肾细胞293T，Western印迹鉴定RBPMS的表达，并利用激光共聚焦显微镜观察RBPMS不同剪接体在细胞中的定位。结果：限制性内切酶分析和DNA序列测定表明构建的重组表达载体正确，Western印迹实验证明RBP1～RBP4表达成功。通过激光共聚焦显微镜观察，RBP1／4围绕胞核在核膜的周围呈聚集状分布；RBP2则在细胞质和细胞核中均有分布，但会出现斑点状聚集；RBP3呈半月状紧密分布在细胞核周围；RBPMS中的RNA识别基序缺失后，这种现象消失，与空载体对照类似，在细胞核和细胞质中均有分布。结论：构建并表达了RBPMS基因的真核表达载体，RBPMS不同剪接体及RNA识别基序缺失后具有不同的亚细胞分布模式，提示具有不同的功能。

Objective： To construct the eukaryotic expression vectors of different RNA binding protein with multi- ple splicing（RBPMS） isoforms, express the RBPMS isoform proteins in eukaryotie cells and investigate the subcel- lular localization of RBPMS proteins. Methods： The complete coding sequences of different RBPMS isoforms （named RBPI～RBP4） were amplified by PCR technique from human ovary eDNA library. The products of PCR were inserted into the pEGFP-C1 vector. Human embryo kidney 293T cells were transfected with the recombinant plasmids and the expression of RBPMS isoforms was identified by Western blot. Subcellular distribution of differ- ent RBPMS variants in transfected 293T cells was observed by confocal laser microscope. Results： The expression vectors of different RBPMS isoforms were constructed and confirmed by restriction enzyme digestion and DNA se- quence analysis. Different RBPMS isoforms were expressed in 293T cells. Different subcellular distribution profiles were observed under confocal laser microscope for RBP1/4, RBP2 and RBP3. RBP1/4 was observed around the nu- cleus with the aggregation-like distribution; RBP2 distributed both in the cytoplasm and the nucleus with speckled aggregation; RBP3 was closely distributed in the perinuclear and the phenomenon disappeared when the RNA rec- ognition motif was lack, which was similar to the empty vector control, with the distribution both in the nucleus and cytoplasm. Conclusion： The eukaryotic expression vectors of different RBPMS isoforms were constructed suc- cessfully. Different subcellular distribution profiles of RBPMS isoforms might be related to their different functions.