结核分枝杆菌RD区蛋白EspK的表达及对巨噬细胞Ⅰ型干扰素产生的影响  

Prokaryotic expression of Mycobacterium tuberculosis RD protein EspK and its effect on type Ⅰ interferon production in macrophages

作　　者：钟燕霄 刘丽莎 刘敏[1] 潘勤[1] 章晓联[1] 罗凤玲[1] ZHONG Yanxiao;LIU Lisha;LIU Min;PAN Qin;ZHANG Xiaolian;LUO Fengling(Dept.of Immunology,Wuhan University School of Basic Medical Sciences,Wuhan 430071,Hubei,China)

机构地区：[1]武汉大学基础医学院免疫学系,湖北武汉430071

出　　处：《武汉大学学报（医学版）》2021年第5期743-751,共9页Medical Journal of Wuhan University

基　　金：国家自然科学基金资助项目(编号:81000714);上海市结核病(肺)重点实验室开放基金(2019)。

摘　　要：目的:构建结核分枝杆菌Rv3879c基因的原核表达质粒并进行体外表达,研究Rv3879c基因编码蛋白EspK的生物学功能。方法:将Rv3879c基因克隆至原核表达载体pET-28a中,诱导表达后采用亲和层析纯化重组蛋白,经SDS-PAGE和Western Blot鉴定后,用纯化的蛋白免疫小鼠制备血清多克隆抗体,ELISA法检测免疫小鼠的特异性抗体滴度;体外实验检测EspK蛋白对巨噬细胞Ⅰ型干扰素产生的影响;pulldown实验和质谱筛选宿主细胞内与EspK相互作用的蛋白;运用生物信息学软件ProtParam对蛋白进行理化性质和亲疏水性分析,TMHMM和SignalP 5.0 server预测蛋白的跨膜区和信号肽,NPS@SOPMA预测蛋白的二级结构,Scansite 4.0预测蛋白模体结构。结果:成功构建Rv3879c的重组原核表达质粒;SDS-PAGE和Western Blot结果表明,表达产物亲和层析纯化后,获得分子质量为74 kU的EspK蛋白;ELISA检测免疫小鼠血清抗体滴度可达1∶409 600;EspK蛋白能促进小鼠巨噬细胞Ⅰ型干扰素的表达;成功筛选到4种与EspK相互作用的蛋白;生物信息学分析结果显示EspK蛋白为亲水性蛋白,无跨膜区及信号肽结构;EspK的二级结构主要由α-螺旋和无规则卷曲组成,有两个Erk1模体结构,可能参与多种生物学过程。结论:成功表达并纯化出重组蛋白EspK,EspK蛋白可促进巨噬细胞Ⅰ型干扰素的表达,利用生物信息学分析蛋白的结构及性质,为进一步研究EspK蛋白在结核病发生发展中的作用奠定了基础。Objective: To express the Mycobacterium tuberculosis RD protein EspK by constructing prokaryotic expression plasmid and analyze its biological function. Methods: The Rv3879 c gene was amplified from the genomic DNA of Mycobacterium tuberculosis strain H37 Rv by PCR, the Rv3879 c gene was cloned into the prokaryotic expression vector pET-28 a. The induced recombinant EspK protein was purified by affinity chromatography and identified by SDS-PAGE and Western Blot. To gain the polyclonal antibody of EspK, the mice were immunized with purified protein, and the antibody titer was evaluated by ELISA. The effect of EspK on the production of type Ⅰ interferon in macrophages was detected by qPCR and ELISA. Pulldown experiment and mass spectrometry were used to screen the proteins interacting with EspK. The physicochemical characteristics and hydrophobicity of EspK was analyzed by ProtParam, the transmembrane region and signal peptide were predicted by TMHMM and SignalP5. 0 server, the secondary structure was predicted by NPS@SOPMA, and the motif was predicted by Scancite 4. 0. Results: The prokaryotic expression plasmid carrying Rv3879 c gene was constructed successfully and the protein could be expressed under the induction of IPTG.SDS-PAGE and Western Blot results confirmed that purified EspK(74 kU) was obtained. ELISA results indicated that the titer of EspK polyclonal antibody in immunized mice serum was about1∶409 600. EpsK can promote the expression of interferon-β(IFN-β) in mouse macrophages. Pulldown experiment and mass spectrometry analysis found four proteins may interact with EpsK. EpsK was a hydrophilic protein with two ERK1 motifs, without transmembrane region or signal peptide. EspK mainly consisted of α-helixes and random coils. Conclusion: EspK was expressed and purified successfully, which could induce specific polyclonal antibody in mice. EspK could induce IFN-β in macrophages. Bioinformatics predicted the structure and characteristics of EspK protein. These findings provide a basis for further r

关 键 词：结核分枝杆菌 Rv3879c基因 EspK蛋白 原核表达 Ⅰ型干扰素 

分 类 号：R378.911[医药卫生—病原生物学]