RIG-I蛋白2CARD结构域真核表达载体的构建及其在HeLa细胞中的表达定位

Construction of pCMV-Myc-RIG-I-2CARD vector and its expression and localization in HeLa cells

目的构建RIG-I蛋白2CARD结构域真核表达载体及其在He La细胞中的表达定位。方法RIG-I是一种模式识别受体,它能够参与由MAVS介导的NF-κB天然抗病毒免疫反应。2CARD(Caspase activation and recruitment domain,CARD)结构域是视黄酸诱导基因-I(Retinoin acid inducible gene I,RIG-I)N端的2个串联重复的半胱天冬酶激活招募区,在病毒侵染细胞过程中,2CARD结构域发挥了识别并启动机体免疫应答的功能。本研究通过PCR获取2CARD片段酶切后,与p CMV-Myc载体连接后转染He La细胞,在He La细胞中表达,并应用Western Blot和免疫荧光进行鉴定。结果 2CARD基因在He La细胞中成功表达。结论本研究成功构建了p CMV-Myc-RIG-I-2CARD真核表达载体,通过将RIG-I分子中负责信号转导的2CARD在He La中表达,为研究病毒与RIG-I的互作关系奠定一定的基础。

Objective To construct pCMV-Myc-RIG-I-2CARD vector and give its expression and localization in He La cells.Methods Retinoic acid inducible gene-I（ RIG-I） as the pattern recognition receptor,can participate in the MAVS-mediated NF-κB innate anti-viral immune response. Caspase activation and recruitment domain（ CARD） is two repeatable caspase activation recruiting areas on the N-terminal of RIG-I. In the processes virus infection,2CARD domain plays important roles in recognizing and priming immune response.In this study,2CARD gene was amplified with PCR method,and then was inserted into pCMV-Myc plasmid. The obtained pCMV- Myc-RIG-I-2CARD was transfected into He La cells,and the expression of 2CARD gene was confirmed by Western Blot and immunofluorescent. Results The result showed that the 2CARD fusion protein was successfully expressed in He La cells. Conclusions This research constructs the pCMV-Myc-RIG-I-2CARD vector,through 2CARD fusion protein successfully expressed in He La cells,establishes a foundation to further studies of interactions between virus and RIG-I.