带Flag标签的RNF26真核表达载体构建及功能验证  

Construction of a eukaryotic expression vector of RNF26 and function detection

作　　者：贾松浩 丛瑞 朱祥 霍楠 马路园 唐枝 孙志佳 亢小峰 初众 徐小洁 李荣宽[1] JIA Song-hao;CNG Rui;ZHU Xiang;HUNan;MA Lu-yuan;TANG Zhi;SUN Zhi-jia;KANG Xiao-feng;CHU Zhong;XU Xiao-jie;LI Rong-kuan(l.The Second Hospital of Dalian Medical University,Dalian,Liaoning 116027,China;Institute of Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]大连医科大学附属第二医院,辽宁大连116027 [2]军事科学院军事医学研究院生物工程研究所,北京100850

出　　处：《军事医学》2022年第3期184-187,共4页Military Medical Sciences

基　　金：国家自然科学基金面上项目(81672602)。

摘　　要：目的构建带Flag标签的RING结构泛素连接酶26(Ring finger protein 26,RNF26)真核表达载体,表达Flag-RNF26的融合蛋白,通过细胞免疫荧光实验,观察其在人肝癌细胞HepG2中的定位及与带绿色荧光蛋白标签的死骨片1(sequestosome 1,SQSTM1)是否存在共定位。方法以人乳腺文库为模板,应用PCR技术扩增出RNF26的编码区序列,双酶切后插入带Flag标签的pCMV-Tag2B载体中,构建pCMV-Tag2B-RNF26重组质粒;将测序正确的重组质粒和pCMV-Tag2B空载体分别转染人胚肾293T细胞中,Western印迹检测融合蛋白表达情况,将成功表达的质粒pCMV-Tag2B-RNF26与pEGFP-SQSTM1质粒转染至人肝癌细胞HepG2中,进行免疫荧光染色,荧光显微镜观察SQSTM1和RNF26的共定位情况。结果插入片段的DNA序列测序结果与目的基因序列相同,Western印迹检测提示,融合蛋白成功表达;免疫荧光结果表明,RNF26与SQSTM1蛋白在细胞质中存在共定位。结论pCMVTag2B-RNF26真核表达载体构建成功。带Flag标签的人RNF26蛋白在HepG2细胞内成功表达,且与SQSTM1蛋白存在共定位,为进一步研究RNF26和SQSTM1的相互作用关系建立了实验基础。Objective To construct a eukaryotic expression vector of Ring finger protein 26(RNF26)with Flag tag,express the fusion protein of Flag-RNF26,and observe the localization of RNF26 in human liver cancer cell HepG2and the relationship with SQSTM1 labelled with green fluorescent protein.Methods Using the human breast library as the template,the coding region sequence of RNF26 was amplified by PCR.After double enzyme digestion,the gene segment was inserted into the pCMV-Tag2B vector to construct a pCMV-Tag2B-RNF26 recombinant plasmid.The correctly sequenced recombinant plasmid and pCMV-Tag2B empty vector were transfected into human embryonic kidney 293T cells respectively,and protein expressions were identified by Western blotting.After that,pCMV-Tag2B-RNF26 and pEGFPSQSTM1 were transfected into human hepatoma cell HepG2 for immunofluorescence staining.The co-localization of SQSTM1 and RNF26 was observed under a fluorescence microscope.Results The DNA sequence of the inserted fragment was correct.Western blotting showed that the fusion protein was properly expressed.Immunofluorescence showed that RNF26 and SQSTM1 protein were co-located in the cytoplasm.Conclusion A eukaryotic expression vector pCMVTag2B-RNF26 is constructed.Human RNF26 protein with Flag tag is successfully expressed in HepG2 cells and co-located with SQSTM1 protein.

关 键 词：RNF26 死骨片蛋白1 真核表达 HEPG2细胞 荧光免疫测定 克隆 

分 类 号：R735.7[医药卫生—肿瘤] R446.6[医药卫生—临床医学]