NLRP3基因真核表达载体的构建和定位

Construction of eukaryotic plasmid of NLRP3 gene and the localization of fusion protein

目的:构建人NLRP3真核表达载体,并证实融合蛋白在人心肌细胞中定位。方法:以人胎脑cDNA文库为模版,PCR扩增NLRP3全长编码基因,亚克隆至p3×FLAG-cmvTM-10表达载体中。将构建的重组质粒测序并转染到人心肌细胞中,提取细胞蛋白进行Western blotting检测。利用激光共聚焦扫描显微镜观察FLAG-NLRP3在HCM细胞内定位。结果:NLRP3全长基因序列克隆到了真核表达载体p3×FLAG-cmvTM-10中,酶切鉴定片段大小3 111 bp。Western blotting检测到了融合蛋白FLAG-NLRP3表达,分子量约为114 kD。FLAG-NLRP3在人心肌细胞中表达,定位于细胞浆内。结论:成功构建了FLAG-NLRP3全长基因真核表达载体,FLAG-NLRP3蛋白主要定位于心肌细胞浆内。

Objective： To construct eukaryotic expression plasmid of human NLRP3 gene and identify the localization of recombinant protein in human cardiac myocytes（ HCM）. Methods： The NLRP3 coding sequence was amplified by polymerase chain reaction（ PCR） method from the human fetal brain cDNA library,and was subcloned into p3 × FLAG-cmvTM-10 vector. After the target region was sequenced,the plasmids were transfected into HCM cells,established from HCM. The expression of the recombinant plasmid in HCM cells were proved by Western blotting. The function of FLAG-NLRP3 in HCM was observed by using confocal laser scanning microscopy. Results： NLRP3 had been constructed into expressing vector p3 × FLAG-cmvTM-10 successfully. The length of the fragment was 3 111 bp,identified by restriction enzymes digestion. The expression of FLAG-NLRP3 fusion protein was detected by Western blotting,with a molecular weight 114 kD. Transient overexpression of FLAG-NLRP3 induced apoptosis in HCM cells. Conclusion： The recombinant FLAG-NLRP3 plasmids are successfully cloned into eukaryotic expressing vector. The FLAG-NLRP3 fusion protein is mainly expressed in the cytoplasma in HCM cells.