肺炎支原体MPN662的抗氧化活性研究  

The antioxidant activities of Mycoplasma pneumoniae MPN662

作　　者：何桂婷 李婷婷 张乃宇 唐睿文 刘婷婷 马黎灿 田巍 朱翠明 He Guiting;Li Tingting;Zhang Naiyu;Tang Ruiwen;Liu Tingting;Ma Lican;Tian Wei;Zhu Cuiming(Key Laboratory of Prevention and Control of Special Pathogens in Hunan Provincen,Institute of Pathogen Biology,Hengyang Medical College,University of South China,Hengyang 421001,China;Major in Medical Laboratory Technology,Level 2020,Chuanshan College,University of South China,Hengyang 421001,China;Blood Transfusion Department,the Affiliated Nanhua Hospital,Hengyang Medical School,University of South China,Hengyang 421001,China)

机构地区：[1]南华大学衡阳医学院病原生物学研究所,特殊病原体防控湖南省重点实验室,衡阳421001 [2]南华大学船山学院2020级医学检验技术专业,衡阳421001 [3]南华大学衡阳医学院附属南华医院输血科,衡阳421001

出　　处：《中华微生物学和免疫学杂志》2024年第10期853-859,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(31970177);湖南省自然科学基金(2022JJ30543);湖南省大学生创新创业项目(202212650009,S202212650007)。

摘　　要：目的研究肺炎支原体(Mycoplasma pneumoniae,Mp)MPN662的蛋氨酸亚砜(MetO)还原活性并明确酶活性关键位点,探究MPN662对Mp脂质相关膜蛋白(LAMPs)刺激的人髓系白血病单核细胞(THP-1细胞)产生活性氧(ROS)和超氧化物歧化酶(SOD)的调节。方法构建pET28a(+)-mpn662及突变重组质粒pET28a(+)-mpn662-Ser66(第66位Cys突变为Ser)和pET28a(+)-mpn662-Ala66(第66位Cys突变为Ala),诱导表达、鉴定并纯化重组蛋白MPN662(rMPN662)和重组突变蛋白。DTNB法检测rMPN662和重组突变蛋白还原MetO的活性;逆转录实时荧光定量PCR(qRT-PCR)和Western blot分别检测不同浓度过氧化氢(H2O2)或过氧叔丁醇(t-BHP)刺激Mp后mpn662基因的mRNA转录水平以及Mp MPN662蛋白的表达水平。荧光探针(DCFH-DA)和总SOD活性检测试剂盒检测Mp LAMPs刺激的经rMPN662预处理的THP-1细胞的ROS和SOD含量。结果Mp rMPN662能还原MetO,但将其第66位Cys突变为Ser或Ala后,突变蛋白还原MetO的活性明显下降。H2O2和t-BHP刺激Mp后,mpn662基因的mRNA转录水平和MPN662蛋白表达水平显著增高,且呈剂量依赖性。rMPN662能抑制Mp LAMPs刺激的THP-1细胞合成ROS,且能增加SOD的表达。结论Mp MPN662能够还原MetO为Met,Cys66为其活性关键氨基酸。MPN662可抑制Mp LAMPs刺激的THP-1细胞产生ROS,促进细胞合成SOD。ObjectiveTo investigate the antioxidant function of Mycoplasma pneumoniae MPN662 and analyze the key active sites,and to explore the role of MPN662 in the regulation of the production of reactive oxygen species(ROS)and superoxide dismutase(SOD)in THP-1 cells.MethodspET28a(+)-mpn662,recombinant mutant plasmids pET28a(+)-mpn662-Ser 66(the 66 th Cys was mutated to Ser)and pET28a(+)-mpn662-Ala 66(the 66 th Cys was mutated to Ala)were constructed,recombinant proteins rMPN662,rMPN662-Ser 66 and rMPN662-Ala 66 were expressed,identified,and purified.DTNB method was employed to analyze the MetO reduction activity of rMPN662 and recombinant mutant protein.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot were applied to examine the transcription level of the mpn662 gene and the expression level of MPN662 protein after Mycoplasma pneumoniae were stimulated with different concentrations of hydrogen peroxide(H 2O 2)or tert-butyl hydroperoxide(t-BHP),respectively.Fluorescent probes(DCFH-DA)and the total SOD activity detection kit were used to test the levels of intracellular ROS and SOD in THP-1 cells,which were pretreated with rMPN662,and then stimulated by Mycoplasma pneumoniae lipid-associated membrane proteins(LAMPs).Results Mycoplasma pneumoniae rMPN662 could reduce MetO to Met,and the enzyme activities of mutant protein were significantly lower than those of rMPN662 protein.mpn662 gene mRNA transcription level and MPN662 protein expression level were significantly increased in a dose-dependent manner when Mycoplasma pneumoniae was stimulated with H 2O 2 and t-BHP.Treatment with rMPN662 before THP-1 cells were exposed to LAMPs could decrease the level of ROS and increase the production of SOD.Conclusions Mycoplasma pneumoniae MPN662 can reduce MetO to Met,and Cys66 is the key amino acid for this activity.MPN662 can decrease the release of ROS and increase the production of SOD in Mycoplasma pneumoniae LAMPs stimulated THP-1 cells.

关 键 词：肺炎支原体 MPN662 抗氧化活性 活性氧 超氧化物歧化酶 

分 类 号：R375[医药卫生—病原生物学]