人可溶性DSG2胞外域蛋白的真核表达、纯化及活性鉴定  

Eukaryotic expression,purification and activity characterization of human soluble DSG2 extracellular domain protein

作　　者：陈楠 李晓月 顾欣雨 吴同鑫 章汝 李云[1] 唐香平 戴劲 易永祥 CHEN Nan;LI Xiao-yue;GU Xin-yu;WU Tong-xin;ZHANG Ru;LI Yun;TANG Xiang-ping;DAI Jin;YI Yong-xiang(Nanjing Hospital Affiliated to Nanjing University of Chinese Medicine,Nanjing 210003,China;School of Public Health,Nanjing Medical University,Nanjing 211166,China)

机构地区：[1]南京中医药大学附属南京医院,江苏南京210003 [2]南京医科大学公共卫生学院,江苏南京211166

出　　处：《海南医学院学报》2023年第10期721-727,共7页Journal of Hainan Medical University

基　　金：南京市科技计划项目(ZX20200009);江苏省研究生科研与实践创新计划项目(SJCX22‐0895)。

摘　　要：目的:构建DSG2与人IgG Fc片段融合的分泌型真核表达载体,在293T细胞中验证其表达,纯化具有生物活性的分泌型蛋白。方法:通过PCR扩增DSG2胞外域片段基因(DSG2 extracellular domain,DSG2ex),并将其整合到真核表达质粒pCMV3-IgG1中,构建重组真核表达质粒pCMV3-DSG2ex-IgG1。将构建成功的真核表达质粒转染入293T细胞中,使细胞外分泌表达DSG2胞外域蛋白,收集细胞上清液,经亲和层析Protein A纯化后,进行Western Blotting和ELISA活性鉴定。结果:成功构建了pCMV3-DSG2ex-IgG1真核表达质粒。转染后96 h蛋白表达量最高,表达产物纯化后经SDS-PAGE电泳分析其相对分子质量在100~130 kDa之间,与预期一致,纯化的融合蛋白纯度达90%以上,含量为0.8 mg/mL。通过Western Blot检测纯化后带有IgG1标签的DSG2胞外域蛋白能被IgG单克隆抗体识别。ELISA结果显示制备的DSG2胞外域蛋白具有明显结合人55型腺病毒(HAdV-55)Fiber Knob蛋白的活性,结论:成功建立了一种简单高效的人可溶性DSG2胞外域蛋白真核表达及纯化的方法,且纯化出来了具有生物活性的DSG2胞外域蛋白,为后期其蛋白功能研究及抗腺病毒药物的研究奠定了基础。Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified pro-tein reached 0.8 mg/mL with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.

关 键 词：人可溶性DSG2胞外域蛋白 真核表达 纯化 活性鉴定 

分 类 号：Q78[生物学—分子生物学] R914[医药卫生—药物化学]