生殖支原体GroEL蛋白的生物信息学分析、表达纯化及致炎分析  

Bioinformatics,expression,purification,and inflammation-inducing effect of Mycoplasma genitalium GroEL protein

作　　者：陈丽 苏晓玲 罗浩荡 王婧芸 廖道庸 甘甜 于建威 何军[1,2,3] CHEN Li;SU Xiaoling;LUO Haodang;WANG Jingyun;LIAO Daoyong;GAN Tian;YU Jianwei;HE Jun(Department of Laboratory Medicine,Affiliated Nanhua Hospital,University of South China,Hengyang 421001,Hunan,China;Department of Blood Transfusion,Affiliated Nanhua Hospital,University of South China,Hengyang 421001,Hunan,China;Clinical Research Institute,Affiliated Nanhua Hospital,University of South China,Hengyang 421001,Hunan,China)

机构地区：[1]南华大学附属南华医院检验科,湖南衡阳421001 [2]南华大学附属南华医院输血科,湖南衡阳421001 [3]南华大学附属南华医院临床研究所,湖南衡阳421001

出　　处：《生物工程学报》2024年第11期4084-4097,共14页Chinese Journal of Biotechnology

基　　金：湖南省自然科学基金(2023JJ60055,2024JJ7459,2024JJ9397);湖南省卫生健康委卫生科研课题(20201915);湖南省卫生健康高层次人才支持计划(20240304128);衡阳市科技计划(202250045307)。

摘　　要：为了解生殖支原体(Mycoplasma genitalium,Mg)GroEL蛋白的致病机制,本研究运用生物信息学软件对MgGroEL蛋白结构和功能进行预测和分析,构建pET-28a-GroEL重组质粒,使用0.2 mmol/L IPTG诱导蛋白表达,通过Ni-亚氨基二乙酸(iminodicitic acid,IDA)柱亲和纯化获得Mg rGroEL蛋白。将浓度为2μg/mL的Mg rGroEL蛋白作用于人单核细胞白血病THP-1细胞,通过ELISA检测细胞上清液中炎症因子IL-1β和TNF-α的含量,使用全自动化学发光仪检测细胞上清液中炎症因子IL-6的含量,通过细胞免疫荧光和Western blotting观察NF-κB信号通路激活情况。结果表明,Mg GroEL蛋白是含有543个氨基酸的酸性带负电荷蛋白,相对分子质量为58.44 kDa,等电点为5.68,分子式为C_(2568)H_(4300)N_(700)O_(825)S_(8),为稳定的亲水性蛋白;二级结构以α-螺旋和无规则卷曲为主;MgGroEL蛋白存在12个B细胞优势抗原表位和10个T细胞优势抗原表位。与肺炎支原体、无乳支原体、关节炎支原体、猪肺炎支原体和牛支原体的GroEL蛋白高度同源;Mg rGroEL蛋白能激活THP-1细胞NF-κB信号通路并促进IL-1β、IL-6和TNF-α的分泌。这些结果表明Mg GroEL具有较好的抗原性,能诱导宿主细胞发生炎症反应,本研究为进一步研究GroEL蛋白在Mg中的功能及其致病机制奠定了理论基础。To preliminarily understand the pathogenic mechanism of Mycoplasma genitalium(Mg)GroEL protein,we used bioinformatics tools to predict the structure and function of Mg GroEL protein and then constructed the recombinant plasmid pET-28a-GroEL.The protein expression was induced by 0.2 mmol/L IPTG,and the expressed protein was purified by Ni-iminodicitic acid(IDA)column affinity.Tohoku Hospital Pediatrics-1(THP-1)cells were exposed to 2μg/mL Mg rGroEL.The levels of interleukin(IL)-1βand tumor necrosis factor(TNF)-αin the cell supernatant were measured by ELISA,and that of IL-6 was measured by an automatic chemiluminescence instrument.The activation of the nuclear factor-kappa B(NF-κB)signaling pathway was visualized by immunofluorescence and Western blotting.The results showed that Mg GroEL was a stable hydrophilic protein composed of 543 amino acid residues,with the relative molecular mass of 58.44 kDa,an isoelectric point of 5.68,and a molecular formula of C2568H4300N700O825S8.The secondary structure was mainly composed ofα-helices and random coils.Mg GroEL contained 12 B-cell dominant epitopes and 10 T-cell dominant epitopes.It exhibited high homology with the GroEL proteins from Mycoplasma pneumoniae,M.agalactiae,M.arthritidis,M.hyopneumoniae,and M.bovis.Mg rGroEL activated the NF-κB signaling pathway and promoted the secretion of IL-1β,IL-6,and TNF-αin THP-1 cells.These results suggest that Mg GroEL exhibits substantial antigenicity and possesses the capability of triggering inflammation in host cells.This study establishes a theoretical basis for future investigations pertaining to the role and pathogenic mechanisms of Mg GroEL.

关 键 词：生殖支原体 GroEL蛋白 原核表达 生物信息学 炎症 

分 类 号：Q811.4[生物学—生物工程] R375[医药卫生—病原生物学]