人心肌细胞中NLRP3融合蛋白表达的鉴定

Identification of NLRP3 Recombinant Protein Expression in Human Cardiac Myocytes

目的构建人NLRP3真核表达载体并证实NLRP3融合蛋白在人心肌细胞中表达。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pCDNA3.1-myc-his A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 blot检测。利用流式细胞仪观察Myc-NLRP3在人心肌细胞内的作用。结果 NLRP3全长基因序列克隆到了真核表达载体pCDNA3.1-myc-his A中,酶切鉴定片段大小为3 111 bp。Western blot检测到了融合蛋白Myc-NLRP3表达,分子量约为114 KD。Myc-NLRP3在人心肌细胞中过表达,能够促进细胞凋亡。结论成功构建了Myc-NLRP3全长基因真核表达载体,过表达Myc-NLRP3能够促进心肌细胞凋亡。

Objective To construct the eukaryotic expression plasmid of human NLRP 3 gene and identify its recombinant protein expression 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 pCDNA3.1-myc-his A vector.After the target region was sequenced ,the recombinant plasmids were transfected into HCM cells .The expression of the recombinant plasmid in HCM cells were proved by Western blot .The function of Myc-NLRP3 in HCM cells was observed by using flow cytometry .Results NLRP3 had been constructed into expressing vector pCDNA3.1-myc-his A successfully .The length of the fragment was 3 111 bp,identified by restriction enzymes digestion.The expression of Myc-NLRP3 fusion protein was detected by Western blot , with a molecular weight of 114 KD.The transient overexpression of Myc-NLRP3 induced apoptosis in HCM cells .Conclusion The recombinant Myc-NLRP3 plasmids are successfully cloned into eukaryotic expressing vector .The transient overexpression of Myc-NLRP3 induces apoptosis in HCM cells .