Article(id=1308840486610240311, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840287129141340, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2025-1108, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1758124800000, receivedDateStr=2025-09-18, revisedDate=1760284800000, revisedDateStr=2025-10-13, acceptedDate=null, acceptedDateStr=null, onlineDate=1789981489087, onlineDateStr=2026-09-21, pubDate=1775923200000, pubDateStr=2026-04-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1789981489087, onlineIssueDateStr=2026-09-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1789981489087, creator=13701087609, updateTime=1789981489087, updator=13701087609, issue=Issue{id=1308840287129141340, tenantId=1146029695717560320, journalId=1189982191388893191, year='2026', volume='61', issue='4', pageStart='1001', pageEnd='1434', issueExtLink='null', onlineDate='null', pubDate='1775923200000', pubDateStr='2026-04-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1789981441528, creator='13701087609', updateTime=1789984228828, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1308851977992368434, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840287129141340, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1308851977992368435, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308840287129141340, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1276, endPage=1282, ext={EN=ArticleExt(id=1308840486811566904, articleId=1308840486610240311, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=The direct target and synergistic action of sulbactam against Acinetobacter baumannii, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

β-Lactamase inhibitor sulbactam has unique activity against Acinetobacter baumannii, but its direct target protein has not been fully elucidated. In this research, thermal proteome profiling and surface plasmon resonance technologies were applied to identify and validate RNA 5′ pyrophosphohydrolase (RppH) as the direct target of sulbactam against A. baumannii. And further, using the checkerboard method, we found that sulbactam could synergize with carbapenem antibiotics to battle A. baumannii. The research results supplement the new molecular mechanism of action of sulbactam and provide an effective combined regimen for its combination with other antibiotics to combat clinical intractable drug-resistant bacteria infections.

, authors=Xin-yi LI, Qiong-lu DUAN, Xi ZHU, Ya-nan NI, Dan-qing SONG, Ying-hong LI*, authorsList=Xin-yi LI, Qiong-lu DUAN, Xi ZHU, Ya-nan NI, Dan-qing SONG, Ying-hong LI, authorCompany=null, correspAuthors=Ying-hong LI, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2026 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1308840487923057475, articleId=1308840486610240311, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=舒巴坦抗鲍曼不动杆菌直接靶点及协同增敏抗生素的研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

β-内酰胺酶抑制剂舒巴坦具有独特的抗鲍曼不动杆菌活性, 但其直接靶蛋白尚未完全阐明。本研究运用包括热蛋白质组分析、表面等离子共振等化学蛋白组学技术, 发现并验证RNA 5′焦磷酸水解酶为舒巴坦抗鲍曼不动杆菌的直接靶点。并进一步通过棋盘法筛选发现, 舒巴坦可以协同碳青霉烯类抗生素对抗鲍曼不动杆菌的活性。研究结果补充了舒巴坦新的分子作用机制, 并为其配伍对抗临床棘手耐药菌感染提供了有效联合治疗方案。

, authors=李欣怡, 段琼璐, 朱喜, 倪亚楠, 宋丹青, 李迎红*, authorsList=李欣怡, 段琼璐, 朱喜, 倪亚楠, 宋丹青, 李迎红, authorCompany=null, correspAuthors=李迎红, authorNote=

作者贡献:

李欣怡负责实验部分及初稿的撰写; 段琼璐、朱喜和倪亚楠提供了实验方法学上的指导; 宋丹青对实验过程中出现的问题及论文撰写提供了有益的指导; 本文的通讯作者李迎红研究员负责实验设计和把关、稿件修改等工作。

, correspAuthorsNote=
李迎红,Tel: 86-10-63165284, Fax: 86-10-63165268, E-mail:
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MIC used alone/µg·mL-1MIC of SUL /µg·mL-1MICs in combination/µg·mL-1FIC
AntibioticMICAntibioticMIC (MICSUL)
Meropenem8.1Meropenem (SUL)2 (0.125)0.375
Imipenem4.1Imipenem (SUL)1 (0.25)0.5
Faropenem16.1Faropenem (SUL)4 (0.125)0.375
Amikacin4.1Amikacin (SUL)> 16 (0.063)> 2.06
Clarithromycin16.1Clarithromycin (SUL)0.062 5 (0.5)0.504
Azithromycin4.1Azithromycin (SUL)0.25 (0.5)0.56
Ceftazidime16.1Ceftazidime (SUL)4 (0.5)0.75
Tigecycline0.251Tigecycline (SUL)0.5 (0.03)2.03
Minocycline1.1Minocycline (SUL)0.5 (0.015)0.53
Polymyxin B16.1Polymyxin B (SUL)32 (0.063)2.06
Polymyxin E2.1Polymyxin E (SUL)4 (0.125)2.125
Levofloxacin0.51Levofloxacin (SUL)0.03 (0.5)0.56
Ciprofloxacin0.51Ciprofloxacin (SUL)0.5 (0.03)1.03
Linezolid> 1 024.1Linezolid (SUL)1 024 (0.125)< 0.675
), ArticleFig(id=1308840496777233277, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308840486610240311, language=CN, label=Tab.1, caption=

In vitro antibacterial activities of sulbactam combined with 14 antibiotics against A. baumannii. MIC: Minimal inhibitory concentration; FIC: Fractional inhibitory concentration

, figureFileSmall=null, figureFileBig=null, tableContent=
MIC used alone/µg·mL-1MIC of SUL /µg·mL-1MICs in combination/µg·mL-1FIC
AntibioticMICAntibioticMIC (MICSUL)
Meropenem8.1Meropenem (SUL)2 (0.125)0.375
Imipenem4.1Imipenem (SUL)1 (0.25)0.5
Faropenem16.1Faropenem (SUL)4 (0.125)0.375
Amikacin4.1Amikacin (SUL)> 16 (0.063)> 2.06
Clarithromycin16.1Clarithromycin (SUL)0.062 5 (0.5)0.504
Azithromycin4.1Azithromycin (SUL)0.25 (0.5)0.56
Ceftazidime16.1Ceftazidime (SUL)4 (0.5)0.75
Tigecycline0.251Tigecycline (SUL)0.5 (0.03)2.03
Minocycline1.1Minocycline (SUL)0.5 (0.015)0.53
Polymyxin B16.1Polymyxin B (SUL)32 (0.063)2.06
Polymyxin E2.1Polymyxin E (SUL)4 (0.125)2.125
Levofloxacin0.51Levofloxacin (SUL)0.03 (0.5)0.56
Ciprofloxacin0.51Ciprofloxacin (SUL)0.5 (0.03)1.03
Linezolid> 1 024.1Linezolid (SUL)1 024 (0.125)< 0.675
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舒巴坦抗鲍曼不动杆菌直接靶点及协同增敏抗生素的研究
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李欣怡 , 段琼璐 , 朱喜 , 倪亚楠 , 宋丹青 , 李迎红 *
药学学报 | 研究论文 2026,61(4): 1276-1282
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药学学报 |研究论文 2026 , 61 (4) : 1276 -1282
舒巴坦抗鲍曼不动杆菌直接靶点及协同增敏抗生素的研究
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李欣怡, 段琼璐, 朱喜, 倪亚楠, 宋丹青, 李迎红*
作者信息
  • 中国医学科学院 北京协和医学院, 医药生物技术研究所, 北京 100050
通讯作者:
李迎红,Tel: 86-10-63165284, Fax: 86-10-63165268, E-mail:
作者简介:

作者贡献:

李欣怡负责实验部分及初稿的撰写; 段琼璐、朱喜和倪亚楠提供了实验方法学上的指导; 宋丹青对实验过程中出现的问题及论文撰写提供了有益的指导; 本文的通讯作者李迎红研究员负责实验设计和把关、稿件修改等工作。

The direct target and synergistic action of sulbactam against Acinetobacter baumannii
Xin-yi LI, Qiong-lu DUAN, Xi ZHU, Ya-nan NI, Dan-qing SONG, Ying-hong LI*
Affiliations
  • Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
出版时间: 2026-04-12 doi: 10.16438/j.0513-4870.2025-1108
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β-内酰胺酶抑制剂舒巴坦具有独特的抗鲍曼不动杆菌活性, 但其直接靶蛋白尚未完全阐明。本研究运用包括热蛋白质组分析、表面等离子共振等化学蛋白组学技术, 发现并验证RNA 5′焦磷酸水解酶为舒巴坦抗鲍曼不动杆菌的直接靶点。并进一步通过棋盘法筛选发现, 舒巴坦可以协同碳青霉烯类抗生素对抗鲍曼不动杆菌的活性。研究结果补充了舒巴坦新的分子作用机制, 并为其配伍对抗临床棘手耐药菌感染提供了有效联合治疗方案。

鲍曼不动杆菌  /  舒巴坦  /  热蛋白质组分析  /  RNA 5′焦磷酸水解酶  /  棋盘法

β-Lactamase inhibitor sulbactam has unique activity against Acinetobacter baumannii, but its direct target protein has not been fully elucidated. In this research, thermal proteome profiling and surface plasmon resonance technologies were applied to identify and validate RNA 5′ pyrophosphohydrolase (RppH) as the direct target of sulbactam against A. baumannii. And further, using the checkerboard method, we found that sulbactam could synergize with carbapenem antibiotics to battle A. baumannii. The research results supplement the new molecular mechanism of action of sulbactam and provide an effective combined regimen for its combination with other antibiotics to combat clinical intractable drug-resistant bacteria infections.

A. baumannii  /  sulbactam  /  thermal proteome profiling  /  RNA 5′ pyrophosphohydrolase  /  checkerboard method
李欣怡, 段琼璐, 朱喜, 倪亚楠, 宋丹青, 李迎红. 舒巴坦抗鲍曼不动杆菌直接靶点及协同增敏抗生素的研究. 药学学报, 2026 , 61 (4) : 1276 -1282 . DOI: 10.16438/j.0513-4870.2025-1108
Xin-yi LI, Qiong-lu DUAN, Xi ZHU, Ya-nan NI, Dan-qing SONG, Ying-hong LI. The direct target and synergistic action of sulbactam against Acinetobacter baumannii[J]. Acta Pharmaceutica Sinica, 2026 , 61 (4) : 1276 -1282 . DOI: 10.16438/j.0513-4870.2025-1108
鲍曼不动杆菌(A. baumannii) 是超级细菌“ESKAPE”成员之一, 主要影响危重症患者等免疫力低下人群, 常通过院内感染途径传播[1]。在全球范围内, A. baumannii每年造成数百万人感染, 尤其耐碳青霉烯类鲍曼不动杆菌(carbapenem-resistant A. baumannii, CRAB) 等耐药菌株感染的相关死亡率高达70%[2]。2024年世界卫生组织(WHO) 发布的细菌重点病原体清单中, CRAB被列为“关键优先级”病原体, 即对公共卫生构成最严重威胁的抗生素耐药细菌[3]。我国是CRAB感染的重灾区, 自2005年至2024年, A. baumannii对碳青霉烯类药物的耐药率已从约40%攀升至近75%[4]。因此, 如何有效应对A. baumannii感染, 特别是CRAB感染, 已成为全球公共卫生领域亟待解决的难题。
β-内酰胺酶抑制剂舒巴坦(sulbactam, 图1) 与β-内酰胺类抗生素联合使用, 首个舒巴坦/氨苄西林复方1987年获批上市[5]。此后, 不同配伍的多个复方在临床上被广泛用于治疗多种细菌感染[6]。进一步研究证实, 舒巴坦本身对A. baumannii也显示较好活性[7]。但另一种β-内酰胺酶抑制剂他唑巴坦(tazobactam, 图1) 结构与舒巴坦类似, 却对A. baumannii无抗菌活性[8]。这一差异提示, 舒巴坦的抗菌作用可能不依赖于其对β-内酰胺酶的抑制能力。机制研究表明, 舒巴坦可能通过CarO β-桶蛋白通道进入细菌细胞内[9], 并以共价方式不可逆地结合青霉素结合蛋白PBP3和PBP1a, 从而发挥抗A. baumannii作用[10]。然而, 舒巴坦与PBP3和PBP1a的较弱的亲和力尚不足以完全解释其显著的抗菌活性[10]。Lin等[11]近期发现, 舒巴坦能够显著下调多种与蛋白质合成密切相关的蛋白表达, 包括ATP结合盒转运蛋白及30S和50S核糖体亚基蛋白。但舒巴坦的直接靶点及键合位点等化学生物学作用机制有待进一步阐明。基于此, 本研究拟采用热蛋白质组学分析(thermal proteome profiling, TPP) 等技术, 鉴定舒巴坦在A. baumannii中的作用直接靶蛋白, 为其抗A. baumannii的临床应用提供科学数据。
舒巴坦与β-内酰胺酶抑制剂杜洛巴坦(durlobactam, 图1) 的合剂于2025年获批上市[12]。同时, 舒巴坦与多黏菌素类、四环素类、β-内酰胺类及喹诺酮类抗生素的联用亦有报道, 但不同研究间存在显著异质性[13]。因此, 本论文采用棋盘法, 系统筛选了舒巴坦与不同类型抗生素协同抗A. baumannii的活性, 探索可增敏舒巴坦的抗生素组合及其协同作用广度。
本研究主要采用TPP技术鉴定舒巴坦作用于A. baumannii的靶蛋白, 进而揭示药物作用靶点及蛋白质相互作用的动态网络[14] (图2A)。
以A. baumannii ATCC 19606裂解液为研究样本, 检测60 ℃加舒巴坦组(A)、30 ℃加舒巴坦组(B) 及60 ℃不加舒巴坦组(C) 的上清蛋白。TPP结果显示, 样本的主成分分析(principal components analysis, PCA) 与相关性分析结果分别如图2B、C所示。其中, 第一主成分(principal components 1, PC1) 为原始数据中方差最大的线性组合, 第二主成分(principal components 2, PC2) 为与PC1正交方向上方差最大的线性组合(图2B)。A、B、C组样本的散点在组内呈聚集分布, 表明组内重复性良好; 且PC1贡献率达70.2%, 提示3组间具有较好的区分度。相关性分析(图2C) 显示, 组间整体呈正相关, 同组样本差异较小而组间差异较大, 表明组间样本相对独立。聚类热图结果显示, 相较C组, A组超过半数蛋白质表达下调, 且表达变化趋势呈明显聚类特征, 可归为2个聚类(图2D)。结果提示, 实验组与对照组存在显著差异, 即舒巴坦对A. baumannii的作用可被明确观测到, 为后续结果分析提供了基础。
进一步基于蛋白质的显著性水平和差异倍数绘制火山图(图3A), 图中红色代表上调蛋白, 蓝色代表下调蛋白, 颜色更深表示差异更显著, 横纵坐标值距离0点越远分别对应差异性显著性越高与差异倍数越大。本研究以P < 0.05和FC (Foldchange) ≥ 2.0作为差异蛋白的筛选标准, 对比A组与C组, 共筛选出921个差异表达蛋白, 其中308个呈上调表达, 613个呈下调表达。优先对上调差异表达蛋白按FC值排序分析, 结果显示: 相较于C组, A组中的GTP结合蛋白(probable GTP-binding protein EngB, EngB)、跨膜蛋白(transmembrane protein, TP) 和RNA焦磷酸水解酶(RNA 5′ pyrophosphohydrolase, RppH) 的表达显著上调, FC值分别为达267.06、34.54和34.41。A组与B组对比, EngB表达明显上调(FC值为65.20), 而TP和RppH变化不大, 提示EngB可能不是舒巴坦作用的直接靶点。
KEGG富集分析结果(图3B) 显示, A组与C组之间大部分上调的差异表达蛋白显著富集于代谢相关通路(如苯甲酸盐代谢、氧化磷酸化) 及遗传信息处理相关通路(如核糖体、RNA降解), 且上述通路均具有统计学意义(P < 0.05)。
TP主要是在细胞的跨膜物质转运、能量转化、信号转导、膜稳态维持等方面发挥作用[15], 尽管TP表达呈显著上调, 但相关信号通路的整体上调幅度并不明显, 且其实际涉及的作用通路与KEGG富集分析中显示的显著差异通路结果不一致。RppH主导mRNA降解, 通过特异性去除mRNA 5′端的焦磷酸基团, 将5′端的三磷酸或二磷酸结构转化为单磷酸, 从而启动mRNA的降解[16]。这一机制参与调控遗传信息处理过程, 与KEGG富集分析结果相一致。
通过STRING数据库考察差异表达蛋白质之间的相互作用, 并以此构建了蛋白质互作网络(图3C)。RppH处于该网络的核心位置, 其表达变化显著, 表现为log2 (FC) 值较高, 且在与其他蛋白质的互作中发挥关键节点作用。以上结果提示, RppH蛋白可能是舒巴坦发挥抗A. baumannii作用的关键靶蛋白。
为了进一步确证舒巴坦与RppH的直接相互作用, 本研究采用Discovery Studio 4.5软件对舒巴坦与RppH (PDB ID: 2KDV) 进行了CDOCKER分子对接试验, -CDOCKER ENERGY打分为7.89 kcal·mol-1。2D和3D结果(图4A) 显示, 舒巴坦可与RppH活性位点的Lys140、Gln34、Arg27形成氢键, 以上结果提示舒巴坦与RppH间存在较好的相互作用。
分子动力学模拟结果显示(图4B), 舒巴坦与RppH复合物构象的均方根偏差(root mean square deviation, RMSD) 始终保持在 < 4 Å的水平, 且50 ns后趋于稳定, 提示了舒巴坦与RppH结合复合物的稳定性较高。SPR试验结果(图4C) 表明, 舒巴坦结合RppH的强度呈现明显剂量依赖性, KD值为2.81 μmol·L-1, 提示舒巴坦与RppH之间存在较强的亲和力。以上结果提示RppH可能是舒巴坦发挥抗A. baumannii作用的直接靶点之一。据此推测, 舒巴坦可能通过与RppH发生相互作用, 促进细菌内mRNA降解, 进而菌体中30S和50S核糖体亚基蛋白水平显著下调, 最终导致A. baumannii死亡, 与Lin等[11]前期研究发现相吻合。
本研究采用棋盘筛选法[17]系统探索了临床常用的8类14种代表性抗生素, 包括碳青霉烯类、头孢菌素类、氨基糖苷类、大环内酯类、四环素类、多黏菌素类、喹诺酮类和噁唑烷酮类, 与舒巴坦联用对A. baumannii ATCC 19606的抗菌作用。分别测定了抗生素单药、抗生素与舒巴坦联合用药的最低抑菌浓度(minimal inhibitory concentration, MIC) 值, 再根据联合用药最佳效应组合对应的药物MIC值, 计算得到分级抑菌浓度(fractional inhibitory concentration, FIC) 值(表1)。
结果显示, 多数抗生素与舒巴坦联用后, 其MIC值均呈现不同程度的下降。其中, 碳青霉烯类抗生素美罗培南、法罗培南和亚胺培南与舒巴坦联用时, 均呈现协同抗A. baumannii的作用, FIC ≤ 0.5, 美罗培南和法罗培南的协同作用更佳(图5)。黏菌素类与舒巴坦联用显示拮抗作用, 可能由于黏菌素类的阳离子电荷与舒巴坦中的羧基负离子键合, 进而导致黏菌素类的抗菌效果减弱。
这一现象可能与舒巴坦通过与RppH相互作用, 导致A. baumannii外膜蛋白表达量降低, 进而使菌体外膜通透性显著增加有关, 与文献[18]报道的舒巴坦可破坏部分革兰阴性菌外膜结构的结论相一致。
本文采用TPP技术针对舒巴坦抗A. baumannii的直接靶蛋白开展了探索, 通过差异表达蛋白筛选及通路富集分析, 初步确定RppH为舒巴坦的潜在作用靶点; 进一步通过分子对接、动力学模拟以及SPR实验验证二者结合模式并测定结合亲和力, 为阐明舒巴坦的抗A. baumannii的机制提供了潜在靶点。同时, 采用棋盘法筛选, 发现碳青霉烯类抗生素可增敏舒巴坦抗A. baumannii的活性。并进一步分析了舒巴坦通过靶向RppH增加菌体外膜通透性, 进而发挥增敏作用的机制。本研究不仅揭示了RppH作为舒巴坦作用靶点的全新机制以及RppH介导的外膜通透性调控在细菌耐药进化中的潜在作用, 丰富了革兰阴性菌耐药机制的理论体系。同时, 该研究还为应对当前极具挑战性的CRAB感染提供了可行的联合治疗策略, 为延缓耐药危机、延长现有抗生素生命周期提供了重要的科学依据。
本研究按照美国临床和实验室标准协会(Clinical and Laboratory Standards Institute, CLSI) 标准, 采用微量肉汤稀释法进行抑菌活性测定和棋盘法试验。舒巴坦用阳离子调节的Mueller-Hinton肉汤(CAMH肉汤, 含有20~25 mg·mL-1 Mg2+, 10~12.5 mg·mL-1 Ca2+) 在96孔板上沿纵坐标2倍稀释, 抗生素以同样方式沿横坐标稀释, 每孔终体积为100 μL。测试菌株A. baumannii ATCC 19606在37 ℃的Luria-Bertani肉汤(LB肉汤) 中培养至OD值介于0.5~0.8。将菌液调至0.5 McFarland (1.5×108 CFU·mL-1), 并用CAMH肉汤以1∶30稀释至5×106 CFU·mL-1。然后将10 μL细菌悬浮液加入各孔中, 使最终细菌浊度为1×105 CFU·mL-1。将96孔板置于37 ℃恒温孵育20 h。观察菌株生长情况, 测定舒巴坦与抗生素的最低抑菌浓度并计算FIC值, FIC = MIC (A in combination with B)/MIC (A alone) + MIC (B in combination with A)/MIC (B alone), FIC ≤ 0.5时, 两药作用为协同; FIC值介于0.5~1时, 两药作用为相加; FIC值介于1~2, 为无关作用; FIC > 2, 为拮抗作用。
将A. baumannii ATCC 19606菌落接种至含有LB培养基的摇菌管中, 在37 ℃、转速220 r·min-1的条件下孵育至对数期, 菌液离心收集菌体后, 用PBS将菌液沉淀重悬洗涤2~3次, 后按照BL1586A细菌蛋白提取试剂盒(Biosharp, 中国) 收集蛋白上清液并采用Bradford法进行蛋白定量。将舒巴坦(终浓度1 µg·mL-1) 与蛋白上清液分别于60 ℃ (A组) 或30 ℃ (B组) 孵育10 min, 同时设置不加舒巴坦的60 ℃蛋白上清液(组) 作为阴性对照。蛋白质组学定量分析采Pro DIA方法, 并利用高分辨率质谱仪timsTOF HT完成色谱分离与质谱检测(Bruker, 美国)。使用KEGG通路(http://www.genome.jp/kegg/) 数据库对所有鉴定出的蛋白质进行了功能注释, 利用String数据库(https://string-db.org/) 进行了蛋白相互作用分析。
使用Discovery Studio 4.5软件对舒巴坦和RppH进行CDOCKER分子对接实验。使用蛋白质的CHARMM力场和配体的CGenFF力场计算蛋白质和配体之间的相互作用能量作为其评分函数, 将小分子对接到受体结合口袋中, 从而对受体-配体相互作用进行分析, 得到配体-受体相互作用二维平面图及受体结合口袋表面图。
本研究采用GROMACS 2020.6 (MODIFIED版) 开展分子动力学模拟实验。首先选取结合能分数最佳的构象作为动态分析的起始构象, 随后使用Sobtop 1.0 (dev3.1版本) 生成小分子的基础数据文件; 为RppH蛋白分配Amber99_Parmbsc1力场以完成参数准备, 并采用SPC水模型模拟蛋白周围的水环境。为维持系统静电平衡, 实验中添加适量氯化钠作为抗衡离子。在主模拟开始前, 系统需经全面的平衡过程, 以确保模拟系统的稳定性。完成上述准备后, 本研究开展了200 ns的分子动力学模拟运行, 该模拟时长可观察蛋白-配体复合物在较长时间尺度下的行为, 进而深入分析蛋白与配体的动态相互作用及复合物的结合稳定性。
使用Reichert 2SPR进行SPR分析, 测定舒巴坦与RppH作用的亲和力KD值。目标蛋白在pH值为5.0的醋酸钠溶液中, 利用氨基偶联的方式固定在CM芯片(AMETEK Reichert, 美国) 上。所有数据均通过Reichert 2SPR系统软件(AMETEK Reichert, 美国) 中的动力学模型进行分析。

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2026年第61卷第4期
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doi: 10.16438/j.0513-4870.2025-1108
  • 接收时间:2025-09-18
  • 首发时间:2026-09-21
  • 出版时间:2026-04-12
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  • 收稿日期:2025-09-18
  • 修回日期:2025-10-13
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    中国医学科学院 北京协和医学院, 医药生物技术研究所, 北京 100050

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2种不同金属材料的力学参数
科
Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)
属
Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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