NLRP3基因过表达载体的建立及鉴定

Construction and Identification of NLRP3 Gene Over-Expression Vector

本研究旨在构建携带小鼠nlrp3蛋白的过表达真核载体。首先从高脂饮食喂养3个月的C57BL/6J小鼠上剪取肝脏组织,液氮冻存。快速剪取适量肝脏组织,冰上研磨,Trizol法提取总RNA,逆转录获得c DNA,然后PCR扩增nlrp3基因的编码区。PCR产物电泳回收,经过NotⅠ及NheⅠ双酶切后,将其克隆到真核表达载体p CDH-CMV-MCS-EF1-cop GFP-T2A-Puro中。重组质粒电转进入感受态DH5α,过夜培养后挑取单个菌落过夜扩大培养。然后提取质粒,酶切鉴定后测序。将克隆成功的重组质粒转染HEK293T细胞,培养3 d后收集细胞蛋白,用Western blotting检测nlrp3的表达水平,研究显示,重组质粒p CDH-NLRP3较空白质粒p CDH-NULL的nlrp3蛋白表达水平明显增加(p<0.01)。本研究成功构建了nlrp3过表达载体,为进一步研究其作用机制提供了基础工具。

This study aimed to construct an over-expression eukaryotic vector of mouse NLRP3 protein.We first harvested liver tissues from C57BL/6J mice fed with high-fat diet for 3 months and stored them in liquid nitrogen.We quickly cut a proper amount of liver tissue and ground it on ice.Trizol was used to extract total RNA and c DNA was obtained by reverse transcription.Then we amplified coding area of NLRP3 gene by PCR.The product of PCR was conducted gel electrophoresis and recycled.After NotⅠ and NheⅠ double endonucleas digestion,the product was cloned into eukaryotic expression vector p CDH-CMV-MCS-EF1-cop GFP-T2A-Puro.The recombinant plasmid was transfected into DH5α by electroporation.After overnight culture,a single colony was picked and cultured for overnight.Then we extracted plasmid,conducted double endonucleas digestion and DNA sequencing.The successfully cloned recombinant plasmid was transfected into HEK293 T cells,after 3 days of culture,the cell lysates protein was harvested to assess the protein level of NLRP3 by Western blotting.The results showed that the NLRP3 protein expression level of recombinant plasmid p CDH-NLRP3 was significantly higher than that of p CDH-NULL（p〈0.01）.This study successfully constructed a NLRP3 over-expression vector,providing a essential tool for future study of its molecular mechanism.