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Article(id=1190373732196647617, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1190332325088039709, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2025-0208, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1740585600000, receivedDateStr=2025-02-27, revisedDate=1743436800000, revisedDateStr=2025-04-01, acceptedDate=null, acceptedDateStr=null, onlineDate=1761736813829, onlineDateStr=2025-10-29, pubDate=1746979200000, pubDateStr=2025-05-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1761736813829, onlineIssueDateStr=2025-10-29, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1761736813829, creator=13701087609, updateTime=1761736813829, updator=13701087609, issue=Issue{id=1190332325088039709, tenantId=1146029695717560320, journalId=1189982191388893191, year='2025', volume='60', issue='5', pageStart='1183', pageEnd='1572', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1761726941606, creator=13701087609, updateTime=1761813457266, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1190695198163354009, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1190332325088039709, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1190695198163354010, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1190332325088039709, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1272, endPage=1284, ext={EN=ArticleExt(id=1190373732519609031, articleId=1190373732196647617, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Oral engineered microbial biosensor for in vivo ionizing radiation detection, columnId=1190332325767516958, journalTitle=Acta Pharmaceutica Sinica, columnName=Special Reports: Live biotherapeutic products based on engineered bacteria, runingTitle=null, highlight=null, articleAbstract=

Current physical and chemical dosimeters are limited in that they cannot directly measure the biological effects of radiation or detect it within the body. Biosensors based on engineered probiotics demonstrate high stability and safety, can be used to detect ionizing radiation in vivo. In this study, an oral engineered microbial sensor for ionizing radiation detection has been developed. The Escherichia coli Nissle 1917 (EcN) was selected as the chassis strain. Using CRISPR/Cas9 gene-editing technology, the cryptic plasmids of EcN were successfully removed to yield the chassis strain ΔEcN. To design a radiation-responsive gene circuit, the recA promoter from the SOS response pathway was utilized as the radiation response element, while a fluorescent protein served as the reporter element. This system was designed to be induced by mitomycin C and γ-rays. The performance of engineered bacteria with various gene circuits was characterized and optimized, resulting in the selection of improved candidate strains. Animal experiments were approved by the Ethics Committee of Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations (approval number: IACUC-DWZX-2022-521). After comparing the in vivo radiation detection capabilities of these strains, the EC-8, which exhibited higher sensitivity, was identified as the final oral microbial sensor. This research applies synthetic biology principles to design and engineer a probiotic capable of detecting ionizing radiation within the body. The findings offer a novel method for in vivo ionizing radiation detection and lay the foundation for the development of live biotherapeutics for the precise diagnosis of radiation damage.

, correspAuthors=Bo-chuan YUAN, Yi-guang JIN, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2025 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, authorCompany=null, fund=null, authors=null, authorsList=Zi-yuan CHEN, Yun-xia SU, Ya-qian ZHANG, Feng ZHANG, Bo-chuan YUAN, Yi-guang JIN), CN=ArticleExt(id=1190374311228703427, articleId=1190373732196647617, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=检测体内电离辐射的口服工程化微生物传感器研究, columnId=1190332325914317601, journalTitle=药学学报, columnName=专题报道: 基于工程化细菌的活体生物药, runingTitle=null, highlight=null, articleAbstract=

现有物理和化学剂量计不能直接反映电离辐射对生物体的影响, 无法直接在体内检测辐射。以工程益生菌为核心的生物传感器安全、稳定, 可用于电离辐射体内检测。本文构建了用于电离辐射检测的口服工程化微生物传感器, 选用益生型大肠杆菌Escherichia coli Nissle 1917 (EcN) 作为底盘菌株, 通过CRISPR/Cas9基因编辑成功敲除EcN的隐秘质粒并得到ΔEcN。以SOS响应中recA启动子为辐射响应元件, 以荧光蛋白为报告元件设计辐射响应基因线路, 通过丝裂霉素C和γ射线诱导表达, 对不同基因线路的工程菌进行性能表征和优化, 获得优化的候选工程菌株。所有动物实验获得军事科学院军事医学研究院伦理委员会批准且实验均按照相关指导原则和规定进行(批准号: IACUC-DWZX-2022-521)。比较小鼠体内辐射检测性能, 筛选得到灵敏度更高的工程菌EC-8作为口服工程化微生物传感器。本研究利用合成生物学原理设计构建了口服工程益生菌, 虽然目前构建得到的工程益生菌仍存在局限性, 检测特异性及信噪比仍待进一步提高, 但该工程菌可在体内对电离辐射产生响应, 为体内电离辐射剂量检测提供了一种新方法, 有望成为用于辐射损伤精准诊断的活体生物药。

, correspAuthors=袁伯川, 金义光, authorNote=null, correspAuthorsNote=
**袁伯川, E-mail:
金义光, E-mail:
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ACS Synth Biol, 2021, 10: 333-344., articleTitle=null, refAbstract=null)], funds=null, companyList=null, figs=[ArticleFig(id=1190694813352739104, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=EN, label=null, caption=null, figureFileSmall=3uMyTsCwVouyWm1eP2aQbQ==, figureFileBig=Y094MB/cUIsqVU/Qglx7wg==, tableContent=null), ArticleFig(id=1190694813424042274, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=CN, label=Figure 1, caption= The cryptic plasmids curing results. A: PCR verification of pMUT1 and pMUT2 (lane 1 is pMUT1 PCR result of <i>wild type (WT)</i>; lane 2 is pMUT1 PCR result of ΔEcN; lane 3 is pMUT2 PCR result of <i>WT</i>; lane 4 is pMUT2 PCR result of ΔEcN); B: Resistance verification of ΔEcN (from left to right, non-antibiotic plate, streptomycin plate, and kanamycin plate, respectively); C: Nucleic acid electrophoresis of the extracted plasmids (lane 1 is plasmid extraction of <i>WT</i>; lane 2 is plasmid extraction of ΔEcN) , figureFileSmall=3uMyTsCwVouyWm1eP2aQbQ==, figureFileBig=Y094MB/cUIsqVU/Qglx7wg==, tableContent=null), ArticleFig(id=1190694813524705572, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=EN, label=null, caption=null, figureFileSmall=6r04M4l7khZaiuasUKno9g==, figureFileBig=9ht/KSIXfJ9cWO94N62s7Q==, tableContent=null), ArticleFig(id=1190694813939941670, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=CN, label=Figure 2, caption= Schematic diagram of the ionizing radiation-responsive circuit design , figureFileSmall=6r04M4l7khZaiuasUKno9g==, figureFileBig=9ht/KSIXfJ9cWO94N62s7Q==, tableContent=null), ArticleFig(id=1190694814120296744, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=EN, label=null, caption=null, figureFileSmall=P4Wb7jlV3ZdMeEY3S71xnw==, figureFileBig=vnVyzvt982LqALMHF7Gbhw==, tableContent=null), ArticleFig(id=1190694814556504362, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=CN, label=Figure 3, caption= Fluorescence intensity of EC-5 and EC-6 harboring different report elements under different conditions relative fluorescence intensity of EC-5 (A) and EC-6 (B) under different mitomycin C (MMC) concentrations at different recovery time. Relative fluorescence intensity of EC-5 and EC-6 under different MMC concentrations after 6 h recovery (C) and their comparison (D). <i>n</i> = 12, $\bar{x} \pm s$. <sup>***</sup><i>P</i> < 0.001 , figureFileSmall=P4Wb7jlV3ZdMeEY3S71xnw==, figureFileBig=vnVyzvt982LqALMHF7Gbhw==, tableContent=null), ArticleFig(id=1190694814623613228, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=EN, label=null, caption=null, figureFileSmall=me9SNz49qZSzYJ+0KsHVNg==, figureFileBig=eQkfoBT7HzKe/L/lMIHpXA==, tableContent=null), ArticleFig(id=1190694814715887918, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=CN, label=Figure 4, caption= Fluorescence intensity of EC-7 and EC-8 harboring different promoter elements under different conditions. Relative fluorescence intensity of EC-7 (A) and EC-8 (B) under different MMC concentrations at different recovery time. Relative fluorescence intensity of EC-7 and EC-8 under different MMC concentrations after 6 h recovery (C) and comparison of EC-6/7/8 (D). <i>n</i> = 12, $\bar{x} \pm s$. <sup>***</sup><i>P</i> < 0.001 , figureFileSmall=me9SNz49qZSzYJ+0KsHVNg==, figureFileBig=eQkfoBT7HzKe/L/lMIHpXA==, tableContent=null), ArticleFig(id=1190694814841717040, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=EN, label=null, caption=null, figureFileSmall=f68vU/n+mpaJ3MuIxxTc6w==, figureFileBig=kZtau+WMsrclhUZ9OT3MDQ==, tableContent=null), ArticleFig(id=1190694814959157554, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=CN, label=Figure 5, caption= Laser confocal imaging of engineered bacteria before and after MMC induction. A: Images of EC-5 and EC-6 before and after induction, 600 ×, detector gain was 700 V; B: Images of EC-6/7/8 before and after induction, 600 ×, detector gain was 520 V. MMC concentration: 10<sup>-1</sup> μg·mL<sup>-1</sup>; recovery time: 4 h , figureFileSmall=f68vU/n+mpaJ3MuIxxTc6w==, figureFileBig=kZtau+WMsrclhUZ9OT3MDQ==, tableContent=null), ArticleFig(id=1190694815043043636, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=EN, label=null, caption=null, figureFileSmall=089b9zjF56vRe3ikcR8LWg==, figureFileBig=oG0pK5cIQIZ/519hcsaswQ==, tableContent=null), ArticleFig(id=1190694815135318326, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=CN, label=Figure 6, caption= Fluorescence intensity of EC-6 and EC-8 under different conditions. Relative fluorescence intensity of EC-6 (A) and EC-8 (B) under different irradiation dose at different recovery time. Relative fluorescence intensity of EC-6 and EC-8 under different irradiation dose after 6 h recovery (C) and their comparison (D). <i>n</i> = 12,$\bar{x} \pm s$. <sup>***</sup><i>P</i> < 0.001 , figureFileSmall=089b9zjF56vRe3ikcR8LWg==, figureFileBig=oG0pK5cIQIZ/519hcsaswQ==, tableContent=null), ArticleFig(id=1190694815210815800, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=EN, label=null, caption=null, figureFileSmall=uZFXosPGPdaOltTqiXNZLQ==, figureFileBig=f2ILi2a8/WLhkh7iOvN/sQ==, tableContent=null), ArticleFig(id=1190694815533777210, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=CN, label=Figure 7, caption= Fluorescence intensity of EC-6 and EC-8 at different recovery time. Relative fluorescence intensity of EC-6 and EC-8 under 10<sup>-1</sup> μg·mL<sup>-1</sup> MMC induction (A) and 50 Gy irradiation (B) compared with not induced group at different recovery time. <i>n</i> = 12, $\bar{x} \pm s$. <sup>**</sup><i>P</i> < 0.01, <sup>***</sup><i>P</i> < 0.001 , figureFileSmall=uZFXosPGPdaOltTqiXNZLQ==, figureFileBig=f2ILi2a8/WLhkh7iOvN/sQ==, tableContent=null), ArticleFig(id=1190694815697355067, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=EN, label=null, caption=null, figureFileSmall=nfiUaIousifUPwY0WN/smQ==, figureFileBig=ZCz/GH+i6k5dYv6G98ii8g==, tableContent=null), ArticleFig(id=1190694815839961404, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=CN, label=Figure 8, caption= <i>In vivo</i> imaging of intestines and feces of mice after intragastric administration of EC-9. <i>In vivo</i> fluorescence images of intestines (A) and feces (B) of mice at different time points after EC-9 administration; C: The fluorescence intensity analysis of feces. <i>n</i> = 3, $\bar{x} \pm s$ , figureFileSmall=nfiUaIousifUPwY0WN/smQ==, figureFileBig=ZCz/GH+i6k5dYv6G98ii8g==, tableContent=null), ArticleFig(id=1190694816968229182, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=EN, label=null, caption=null, figureFileSmall=DTvqKlEIAn7ZcacBTATEFg==, figureFileBig=LihfVGDyhIJHTgRE2L8VZA==, tableContent=null), ArticleFig(id=1190694817106641216, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=CN, label=Figure 9, caption= <i>In vivo</i> imaging of the intestines and feces of mice administered with different engineered bacteria before and after exposure to radiation. <i>In vivo</i> fluorescence images of intestines (A) and feces (B) of mice administered with different engineered bacteria before and after exposure to radiation; C: The fluorescence intensity analysis of feces, (-) and (+) are represent not irradiated group and irradiated group, respectively. <i>n</i> = 3, $\bar{x} \pm s$. <sup>**</sup><i>P</i> < 0.01, <sup>***</sup><i>P</i> < 0.001 , figureFileSmall=DTvqKlEIAn7ZcacBTATEFg==, figureFileBig=LihfVGDyhIJHTgRE2L8VZA==, tableContent=null), ArticleFig(id=1190694817224081730, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Name Category Source
pEcCas Plasmid Miaoling Biology Co., Ltd.
pEcgRNA Miaoling Biology Co., Ltd.
pT7-sfGFP Miaoling Biology Co., Ltd.
psfGFP Laboratory preserved
pEC-PrecA-eGFP Laboratory preserved
pEC-PrecA-sfGFP This study
pJ23119-EcNBOX-sfGFP This study
pJ23119-SOSBOX-sfGFP This study
DH5α Strain Beijing Biomed Gene technology Co., Ltd.
E. coli Nissle 1917 Donated by Prof. Liu Gang
ΔEcN This study
EC-5 This study
EC-6 This study
EC-7 This study
EC-8 This study
EC-9 This study
), ArticleFig(id=1190694817437991235, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=CN, label=Table 1, caption=

Plasmids and strains used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Name Category Source
pEcCas Plasmid Miaoling Biology Co., Ltd.
pEcgRNA Miaoling Biology Co., Ltd.
pT7-sfGFP Miaoling Biology Co., Ltd.
psfGFP Laboratory preserved
pEC-PrecA-eGFP Laboratory preserved
pEC-PrecA-sfGFP This study
pJ23119-EcNBOX-sfGFP This study
pJ23119-SOSBOX-sfGFP This study
DH5α Strain Beijing Biomed Gene technology Co., Ltd.
E. coli Nissle 1917 Donated by Prof. Liu Gang
ΔEcN This study
EC-5 This study
EC-6 This study
EC-7 This study
EC-8 This study
EC-9 This study
), ArticleFig(id=1190694817572208965, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Name Sequence (5'-3')
pMUT1-check-F AAACTTCCTTCAGGGCTCGGGGAT
pMUT1-check-R AGATTTCATTTAGCCTGCCGCTCG
Muta7 GACCAAGCGATAACCGGATG
Muta8 GTGAGATGATGGCCACGATT
MUT2-sg2-F TAGTATCCCCGAGCCCTGAAGGAA
MUT2-sg2-R AAACTTCCTTCAGGGCTCGGGGAT
RelB-F GCTCTAGACCTGATTATCCAAGGCTCCCAG
pMUT2-RelB-R CCAAGCTTTTATAGCTCATCCAGTGACACCTTTACGCC
His_sfGFP_KpnⅠ GGGGTACCATGGGCAGCAGCCATCATCAT
sfGFP_HindⅢ GCCGCAAGCTTTTATTTGTAGAGCTCAT
SOSBOX_EcN_L CGATTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCTACTGTATGAGCATACAGTAACTAGTTTTGTTTAACTTTAAGAAGGAGAGGTAC
SOSBOX_EcN_S CTCTCCTTCTTAAAGTTAAACAAAACTAGTTACTGTATGCTCATACAGTAGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAAAT
SOSBOX0_L CGATTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCTACTGTATATATATACAGTAACTAGTTTTGTTTAACTTTAAGAAGGAGAGGTAC
SOSBOX0_S CTCTCCTTCTTAAAGTTAAACAAAACTAGTTACTGTATATATATACAGTAGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAAAT
No_SOSBOX_L CGATTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCACTAGTTTTGTTTAACTTTAAGAAGGAGAGGTAC
No_SOSBOX_R CTCTCCTTCTTAAAGTTAAACAAAACTAGTGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAAAT
), ArticleFig(id=1190694817807089991, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1190373732196647617, language=CN, label=Table 2, caption=

Primers used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Name Sequence (5'-3')
pMUT1-check-F AAACTTCCTTCAGGGCTCGGGGAT
pMUT1-check-R AGATTTCATTTAGCCTGCCGCTCG
Muta7 GACCAAGCGATAACCGGATG
Muta8 GTGAGATGATGGCCACGATT
MUT2-sg2-F TAGTATCCCCGAGCCCTGAAGGAA
MUT2-sg2-R AAACTTCCTTCAGGGCTCGGGGAT
RelB-F GCTCTAGACCTGATTATCCAAGGCTCCCAG
pMUT2-RelB-R CCAAGCTTTTATAGCTCATCCAGTGACACCTTTACGCC
His_sfGFP_KpnⅠ GGGGTACCATGGGCAGCAGCCATCATCAT
sfGFP_HindⅢ GCCGCAAGCTTTTATTTGTAGAGCTCAT
SOSBOX_EcN_L CGATTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCTACTGTATGAGCATACAGTAACTAGTTTTGTTTAACTTTAAGAAGGAGAGGTAC
SOSBOX_EcN_S CTCTCCTTCTTAAAGTTAAACAAAACTAGTTACTGTATGCTCATACAGTAGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAAAT
SOSBOX0_L CGATTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCTACTGTATATATATACAGTAACTAGTTTTGTTTAACTTTAAGAAGGAGAGGTAC
SOSBOX0_S CTCTCCTTCTTAAAGTTAAACAAAACTAGTTACTGTATATATATACAGTAGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAAAT
No_SOSBOX_L CGATTTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCACTAGTTTTGTTTAACTTTAAGAAGGAGAGGTAC
No_SOSBOX_R CTCTCCTTCTTAAAGTTAAACAAAACTAGTGCTAGCATTATACCTAGGACTGAGCTAGCTGTCAAAT
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检测体内电离辐射的口服工程化微生物传感器研究
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陈紫垣 , 苏云霞 , 张雅倩 , 张锋 , 袁伯川 * , 金义光 *
药学学报 | 专题报道: 基于工程化细菌的活体生物药 2025,60(5): 1272-1284
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药学学报 | 专题报道: 基于工程化细菌的活体生物药 2025, 60(5): 1272-1284
检测体内电离辐射的口服工程化微生物传感器研究
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陈紫垣, 苏云霞, 张雅倩, 张锋, 袁伯川* , 金义光*
作者信息

通讯作者:

**袁伯川, E-mail:
金义光, E-mail:
Oral engineered microbial biosensor for in vivo ionizing radiation detection
Zi-yuan CHEN, Yun-xia SU, Ya-qian ZHANG, Feng ZHANG, Bo-chuan YUAN* , Yi-guang JIN*
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出版时间: 2025-05-12 doi: 10.16438/j.0513-4870.2025-0208
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摘要
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现有物理和化学剂量计不能直接反映电离辐射对生物体的影响, 无法直接在体内检测辐射。以工程益生菌为核心的生物传感器安全、稳定, 可用于电离辐射体内检测。本文构建了用于电离辐射检测的口服工程化微生物传感器, 选用益生型大肠杆菌Escherichia coli Nissle 1917 (EcN) 作为底盘菌株, 通过CRISPR/Cas9基因编辑成功敲除EcN的隐秘质粒并得到ΔEcN。以SOS响应中recA启动子为辐射响应元件, 以荧光蛋白为报告元件设计辐射响应基因线路, 通过丝裂霉素C和γ射线诱导表达, 对不同基因线路的工程菌进行性能表征和优化, 获得优化的候选工程菌株。所有动物实验获得军事科学院军事医学研究院伦理委员会批准且实验均按照相关指导原则和规定进行(批准号: IACUC-DWZX-2022-521)。比较小鼠体内辐射检测性能, 筛选得到灵敏度更高的工程菌EC-8作为口服工程化微生物传感器。本研究利用合成生物学原理设计构建了口服工程益生菌, 虽然目前构建得到的工程益生菌仍存在局限性, 检测特异性及信噪比仍待进一步提高, 但该工程菌可在体内对电离辐射产生响应, 为体内电离辐射剂量检测提供了一种新方法, 有望成为用于辐射损伤精准诊断的活体生物药。

电离辐射  /  工程益生菌  /  Escherichia coli Nissle 1917  /  微生物全细胞生物传感器  /  活体生物药

Current physical and chemical dosimeters are limited in that they cannot directly measure the biological effects of radiation or detect it within the body. Biosensors based on engineered probiotics demonstrate high stability and safety, can be used to detect ionizing radiation in vivo. In this study, an oral engineered microbial sensor for ionizing radiation detection has been developed. The Escherichia coli Nissle 1917 (EcN) was selected as the chassis strain. Using CRISPR/Cas9 gene-editing technology, the cryptic plasmids of EcN were successfully removed to yield the chassis strain ΔEcN. To design a radiation-responsive gene circuit, the recA promoter from the SOS response pathway was utilized as the radiation response element, while a fluorescent protein served as the reporter element. This system was designed to be induced by mitomycin C and γ-rays. The performance of engineered bacteria with various gene circuits was characterized and optimized, resulting in the selection of improved candidate strains. Animal experiments were approved by the Ethics Committee of Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations (approval number: IACUC-DWZX-2022-521). After comparing the in vivo radiation detection capabilities of these strains, the EC-8, which exhibited higher sensitivity, was identified as the final oral microbial sensor. This research applies synthetic biology principles to design and engineer a probiotic capable of detecting ionizing radiation within the body. The findings offer a novel method for in vivo ionizing radiation detection and lay the foundation for the development of live biotherapeutics for the precise diagnosis of radiation damage.

ionizing radiation  /  engineered probiotic  /  Escherichia coli Nissle 1917  /  microbial whole-cell biosensor  /  live biotherapeutic product
陈紫垣, 苏云霞, 张雅倩, 张锋, 袁伯川, 金义光. 检测体内电离辐射的口服工程化微生物传感器研究. 药学学报, 2025 , 60 (5) : 1272 -1284 . DOI: 10.16438/j.0513-4870.2025-0208
Zi-yuan CHEN, Yun-xia SU, Ya-qian ZHANG, Feng ZHANG, Bo-chuan YUAN, Yi-guang JIN. Oral engineered microbial biosensor for in vivo ionizing radiation detection[J]. Acta Pharmaceutica Sinica, 2025 , 60 (5) : 1272 -1284 . DOI: 10.16438/j.0513-4870.2025-0208
正文
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电离辐射(ionizing radiation, IR) 是指波长短、频率高、能量高的一类电磁波或高能粒子射线, 其携带的能量能够使物质原子或分子中的电子脱离原子核束缚成为自由态[1]。IR包括α、β、γ、X射线及中子辐射等[2], 被广泛用于医疗、农业、废水处理等领域[3-5]。IR对生物体危害较大, 可造成生物体内许多分子的电离, 引起机体内有序的生化反应失衡, 造成细胞、器官和整体水平的严重损伤, 因此其在被广泛应用的同时也引起人们对其安全性的担忧[6]。例如, 肿瘤放疗时采用的电离辐射, 在杀死肿瘤细胞的同时, 也对辐照区域内的正常细胞造成损伤[7]。在福岛和切尔诺贝利事故中, 环境生物经历了严重的电离辐射, 并可能导致基因突变, 严重影响当地居民的生活[8]。因此电离辐射的检测对安全可控地利用各类射线至关重要。
电离辐射的计量单位一般用辐射剂量表示, 单位是戈瑞(Gy) 或希弗(Sv)。目前IR的检测主要依赖各类辐射剂量计, 按照作用原理可分为物理剂量计(如电离室半导体和闪烁体剂量率仪等)[9-11], 以及化学剂量计(如胶片、热释光和凝胶剂量计等)[12-14]。这些检测方法能较精准地测定射线种类和辐射剂量, 但在用于生物体受照剂量时仅作为估算依据, 无法直接反映生物剂量, 且无法在生物体内进行辐射检测, 检测持续性较差。这些缺点限制了它们在肿瘤放疗、特殊作业人群辐射生物剂量评估中的应用, 因此需要开发一种可在体检测人体受照剂量的电离辐射生物检测方法。
肠道是电离辐射的主要靶器官之一[15]。肠道内天然存在约1015数量级的微生物, 组成肠道菌群[16]。近期发现肠道菌群可作为放射性肠损伤标志物[17, 18], 这为以肠道菌为底盘检测人体受照剂量提供了思路。益生菌是肠道中一类对宿主有益的微生物, 具有生物相容性好、安全性高、易于培养的特点, 是当前研制具有检测、诊断、治疗功能的活体生物药的常用底盘[19-21]。目前, 以益生菌为底盘的活体生物药已在疾病检测与治疗方面取得突破性进展。将NO3-传感基因回路导入益生型大肠杆菌Escherichia coli Nissle 1917 (EcN) 中可实现小鼠的肠道炎症监测, 检测限达到微摩尔级(μmol·L-1)[22]。与EcN类似的E. coli K12菌株被构建为可定植肠道监测饮食的工程菌传感器[23]。将S4O64-传感基因回路导入小鼠肠道大肠杆菌中, 实现长达6个月的稳定肠道炎症监测[24]。这些能够检测特定物质的工程化细菌被称为微生物全细胞传感器(microbial whole-cell biosensor, MWCB)[25-27]。以安全的肠道益生菌为底盘的MWCB能够用于体内疾病的检测和诊断, 具有开发成新一代活体生物药的潜力。然而, 目前用于电离辐射检测的工程菌研究较为匮乏, 且多以非益生菌为底盘, 如耐辐射奇球菌和大肠杆菌DH5α等, 均聚焦于实验室场景下的应用, 无法保证体内安全性, 未能实现在生物体内的辐射检测[28, 29]。因此, 以益生菌为底盘研究肠道电离辐射检测工程菌传感器, 实现生物体内的电离辐射检测具有重要意义和应用价值。
EcN安全性高, 易于培养, 常被用于活体生物药研发的底盘菌, 目前该菌株已被开发为药物[30, 31]。本研究选择EcN为底盘菌株, 并对其进行基因改造和优化, 选择细菌DNA损伤相关的SOS响应通路作为辐射传感基本元件, 通过筛选和优化辐射传感基因回路的特定元件, 获得了可应用于生物体内的电离辐射响应肠道工程菌生物传感器。该MWCB能够感知IR发出绿色荧光, 可口服后进入肠道, 在体检测机体受照情况, 通过检测粪便中排出的益生菌荧光可判断机体受照情况。本研究为肠道电离辐射的生物检测提供了新的思路和方法, 为开发在体辐射损伤诊断的活体生物药奠定了基础。
材料与方法
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试剂与耗材    硫酸卡那霉素、硫酸链霉素、氯霉素(K8020、S8290、C8050, 北京索莱宝生物科技有限公司); 琼脂(EZ7890D190, Biofroxx); L-鼠李糖(A65280, 伊诺凯科技有限公司); 蔗糖(20150615, 国药集团化学试剂有限公司); 甘油(GC205008, 武汉赛维尔生物科技有限公司); T4 DNA Ligase、T4 Polynucleotide Kinase Buffer、T4 Polynucleotide Kinase、Q5超保真DNA聚合酶、KpnⅠ、BsaI、ClaⅠ、XbaI (10152495、10119056、10156399、10125119、10206307、10180013、10104969、10219308, New England BioLabs); 2× M5 HiPer plus Taq HiFi PCR mix (22FB0102, 北京聚合美生物科技有限公司); DNA Clean & Concentrator-5 (212352, Zymo Research); 丝裂霉素C (H2211333, 阿拉丁试剂)。本文用水均为去离子水, 使用纯水仪制备。
仪器    60Co γ辐射装置(军事医学研究院); 多功能酶标仪(Spark, 瑞士Tecan公司); 超微量分光光度计(K5600, 北京凯奥科技发展有限公司); 基因导入仪(Xinyi-2E, 宁波新艺超声设备有限公司); 激光共聚焦显微镜(ZEISS LSM 980, 德国Carl Zeiss公司); 多模式动物活体成像系统(Aniview600, 广州博鹭腾生物科技有限公司); 高速组织冷冻研磨仪(KZ-Ⅱ, 武汉赛维尔生物科技有限公司); 纯水仪(Heal Force Super NW, 上海康雷仪器有限公司)。
实验动物    C57小鼠, SPF级, 雄性, 体重20 ± 2 g, 许可证号: SCXK (京) 2021-0006, 斯贝福(北京) 生物技术有限公司。小鼠饲养过程中自由给水进食, 适应环境1周后开始实验。小鼠饲养过程中的饮用水、饲料、饲养环境温度、湿度及光照周期均符合实验动物饲养标准。所有动物实验获得军事科学院军事医学研究院伦理委员会批准且实验均按照相关指导原则和规定进行(批准号: IACUC-DWZX-2022-521)。
质粒与细菌    本研究中所使用的质粒与细菌见表 1。
引物    本研究中所使用的引物见表 2。本文中如无特殊说明, 所使用引物的浓度均为10 μmol·L-1。本文中使用的引物均委托金唯智(GENEWIZ) 生物科技有限公司合成。
电转化感受态细菌的制备    取所需细菌的菌液, 划线接种于LB平板上, 待菌落长出后挑取单菌落, 接种于30 mL的LB液体培养基中震荡培养至吸光度(OD600) 值达到0.3~0.5之间, 于冰上静置10 min后, 于4 ℃、4 000 ×g离心10 min, 弃去上清, 加入20 mL冰上预冷的10%无菌甘油溶液洗涤菌体3次, 再加入1~2 mL冰上预冷的10%无菌甘油溶液轻轻重悬菌体, 分装至1.5 mL离心管中, 每管分装100 μL, 于-80 ℃冰箱中储存。
细菌的电转化    将电击杯置于冰上预冷, 加入100 μL电转化感受态细菌, 随后加入1~5 μL质粒, 于冰上静置10 min, 随后设置电压为2 500 V, 电容为25 μF, 电阻为200 Ω进行电转化。转化完成后将电击杯中的菌液转移至无菌的1.5 mL离心管, 加入0.5 mL的于37 ℃水浴中预热的LB液体培养基震荡培养1.5 h。涂布于相应抗性的LB固体平板, 37 ℃培养箱中倒置培养16 h。
隐秘质粒敲除    参考已有报道进行EcN隐秘质粒敲除[32, 33]。制备Escherichia coli Nissle 1917 (EcN) 的电转化感受态, 将pEcCas质粒转入EcN中, 挑取单克隆扩增培养, 加入终浓度为10 mmol·L-1的鼠李糖诱导Cas9表达。利用CRISPOR在靶基因区域搜索和设计20 bp的单链向导RNA (single-guide RNA, sgRNA) 序列[34], 设计靶向两种隐秘质粒pMUT1/pMUT2的sgRNA序列, 经引物合成、梯度退火、磷酸化得到sgRNA序列, 通过BsaI酶切、ZymocleanTM Gel DNA Recovery Kit胶回收和T4 DNA连接酶连接到pEcgRNA载体上。将两种pEcgRNA质粒逐一转化至EcN, 涂布后挑取单克隆进行PCR验证, 得到隐秘质粒敲除菌株。再将所得菌株接种于在含葡萄糖(5 g·L-1) 的LB液体培养基, 并涂于含有葡萄糖(5 g·L-1) 和蔗糖(10 g·L-1) 的LB平板上, 诱导进行pEcCas和pEcgRNA的清除, 最终获得无质粒菌株ΔEcN。
ΔEcN的基因型验证    对ΔEcN进行扩增培养, 以ΔEcN菌液为模板通过PCR及核酸电泳验证pMUT1与pMUT2。取ΔEcN菌液分别涂布于无抗LB固体平板, 含40 μg·mL-1硫酸卡那霉素的LB固体平板与含60 μg·mL-1链霉素的LB固体平板上, 培养后验证CRISPR/Cas9质粒的清除。最后对ΔEcN进行质粒提取和核酸电泳验证。
传感回路及工程菌的构建    制备ΔEcN电转化感受态细胞, 将pEC-PrecA-eGFP电转化入ΔEcN中, 得到EC-5; 以质粒pT7-sfGFP为模板进行PCR, 通过DNA纯化、KpnⅠ-HF和HindⅢ-HF双酶切和T4连接替换pEC-PrecA-egfp质粒的egfp片段, 得到pEC-PrecA-sfGFP。将质粒电转入ΔEcN中, 命名为EC-6。分别使用引物SOSBOX_EcN_L/SOSBOX_EcN_S与引物SOSBOX0_L/SOSBOX0_S进行退火与磷酸化, 得到启动元件pJ23119-EcNBOX片段和pJ23119-SOSBOX片段。通过KpnⅠ-HF和ClaⅠ双酶切、胶回收和T4连接将两个启动元件分别插入psfGFP质粒中, 得到pJ23119-EcNBOX-sfGFP和pJ23119-SOSBOX-sfGFP。将两个质粒通过电转化的方式转入ΔEcN, 得到EC-7和EC-8。
不同基因回路的传感性能检测    配制1 mg·mL-1的丝裂霉素C (mitomycin C, MMC) 水溶液母液, 通过10倍稀释的方式得到最终浓度为101、100、10-1、10-2、10-3 μg·mL-1的水溶液。分别取EC-5/6/7/8进行扩增培养, 取扩增培养后的菌液, 以不含质粒的ΔEcN为control组。取200 μL菌液加入96孔板中, 每个浓度梯度12个孔中。于37 ℃孵育, 设置不同时间点使用多功能酶标仪检测荧光强度与OD600值, 检测荧光时激发波长为485 nm, 发射波长为535 nm, 每次检测前均振摇5 s。计算相对荧光强度: 相对荧光强度=荧光强度数值(relative fluorescence unit, RFU) / OD600。
工程菌共聚焦显微镜观察    取EC-5/6/7/8工程菌菌液进行培养, 培养后使用10-1 μg·mL-1丝裂霉素C诱导4 h后, 取未诱导与诱导后的菌液各2 μL, 滴在干净的载玻片上, 并盖上盖玻片, 置于共聚焦显微镜下, 于明场与绿色荧光场下分别观察, 并设置ΔEcN作为阴性对照, 放大倍数均为600×。
工程菌辐照响应性能的检测    取EC-6与EC-8进行扩增培养, 取扩增培养后的菌液分装, 每种菌液均分装成7管, 每管分装3 mL菌液, 分别辐照0、10、20、30、40、50、60 Gy, 辐照剂量率为467.36 cGy·min-1。辐照后加入黑色透底96孔板中, 每个辐照梯度加入12个孔, 每个孔均为200 μL菌液。取不含质粒的ΔEcN作为阴性对照。将黑色透底96孔板静置于37 ℃培养箱中进行孵育, 设置不同时间点进行OD600值与荧光强度的检测。
肠道及粪便中工程菌分布的活体成像检测    使用引物No_SOSBOX_L与No_SOSBOX_S进行退火与磷酸化, 使用EC-7构建中得到的骨架载体, 进行T4连接并得到pJ23119-sfGFP, 使用电转化的方法转入ΔEcN中, 命名为EC-9。扩增培养EC-9, 取EC-9菌液100 mL, 离心后使用10 mL的LB培养基重悬, 小鼠灌胃(每只200 μL)。分别取灌胃前、灌胃后即时, 以及灌胃后2、4、6、8、10、12、24、48 h的小鼠肠组织在活体成像仪下观察。活体成像仪曝光时间为0.1 s, 激发滤光片为465 nm, 发射滤光片为540 nm。收集灌胃后0、2、4、6、8、10、12、24 h的小鼠粪便, 于活体成像仪下观察。通过活体成像仪软件AniView划分ROI区并计算信号平均强度。
小鼠辐照方法    取C57小鼠, 随机进行分组, 分为ΔEcN未辐照组、ΔEcN辐照组、EC-6未辐照组、EC-6辐照组、EC-8未辐照组和EC-8辐照组, 每组3只, 给予相应菌液200 μL。辐照组灌胃0.5 h后麻醉进行小鼠腹部局部辐照, 剂量为12 Gy, 剂量率为72.24 cGy·min-1。其他3组不进行辐照。
受照小鼠的肠道及粪便荧光检测    取灌胃后8 h的小鼠肠组织进行活体成像观察。取灌胃后10 h的小鼠粪便进行匀浆, 随后用5 μm无菌过滤器进行过滤。过滤后4 000 ×g、5 min离心, 离心后使用1 mL无菌磷酸盐缓冲液重悬, 将液体加入黑色透底96孔板中, 使用多功能酶标仪按照前述方法进行荧光强度和OD600值的检测。
统计学分析    采用SPSS 16.0软件通过单因素方差分析对数据进行统计学分析。实验数据以均数±标准差($\bar{x} \pm s$) 表示; 比较组间差异, P < 0.05表示具有显著性差异。
结果
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1 底盘菌的优化
野生型EcN含有2个隐秘质粒[35], 导致其遗传转化受到影响。为提高EcN的基因工程化效率, 通过CRISPR-Cas9基因编辑技术敲除了其中的2个隐秘质粒pMUT1和pMUT2, 得到稳定遗传的无质粒ΔEcN菌株, PCR结果、质粒提取结果、抗性验证结果均证明当前菌株不含有任何质粒(图 1)。
2 不同元件对电离辐射传感基因线路检测性能的影响
基因线路是工程菌传感性能的核心。为得到性能较好的基因线路, 本研究分别对报告元件与启动元件进行筛选(图 2)。由于MMC可使细胞的DNA解聚引起DNA损伤, 与电离辐射引起的DNA损伤效应类似, 而MMC处理细菌相比于电离辐射处理更加简便易行, 因此在基因线路筛选评价过程中用MMC处理代替电离辐射照射, 以提高评价效率[36]。
首先, 对报告元件性能进行筛选, 选择两种常用的绿色荧光蛋白eGFP (EC-5) 和sfGFP (EC-6), 通过工程菌诱导后产生的荧光信号相对强度进行比较和筛选, 除整体荧光信号强度外, 还需结合剂量依赖性、灵敏度及信噪比等指标进行综合评价。EC-5的荧光强度弱, 报告效果差, 在MMC诱导浓度大于1 μg·mL-1时, EC-5的荧光值达到平台期, 灵敏度差(图 3A)。EC-6诱导后的荧光强度随时间增强, 最终可达到未诱导组2倍左右的荧光强度, 且EC-6具有剂量依赖性, 在10-3至10-1 μg·mL-1区间内呈现良好线性关系(图 3B)。当MMC剂量过大时, EC-6荧光响应效果变差, 可能是由于剂量过大, 导致细菌死亡率过高导致。复愈6 h后, EC-5诱导组的荧光值和对照组基本无差异甚至低于对照组, 而EC-6响应效果较好, 在MMC浓度为10-3 μg·mL-1时, EC-6即可产生显著的荧光, 在MMC浓度为10-1 μg·mL-1时荧光值最高(图 3C)。在未诱导时, EC-6的荧光响应与control组相比具有明显差异, 推测可能存在荧光蛋白的泄露表达。在MMC浓度为10-3 μg·mL-1时, EC-6的荧光响应强度就显著高于EC-5, EC-6与EC-5相比, 整体的响应强度得到了明显提升(图 3D)。因此, sfGFP与eGFP具有更强的信号和更好的响应能力, 更适合用作报告基因。
随后对启动元件进行筛选。本研究选用的SOS启动元件可分为3个区域, 分别为启动子、核糖体结合位点(ribosome binding site, RBS)、阻遏位点SOSBOX。EC-6的启动元件3个区域均为EcN原有的recA启动子区域天然元件, 在此基础上EC-7将天然启动子替换为组成型强启动子PJ23119, EC-8将启动子、RBS、阻遏位点分别替换为PT7、T7-RBS、优化的SOSBOX。SOSBOX是SOS响应的关键元件, 已有研究表明可对SOSBOX进行人工设计, 可提升SOS响应的性能[37]。EC-6和EC-7的SOSBOX元件为EcN原有的EcNBOX, EC-8则参考文献[37]选择经过人工设计的SOSBOX。
使用MMC对EC-6、EC-7和EC-8进行诱导, 检测3种工程菌的性能以筛选最优启动回路。在复愈2 h后, EC-7与EC-8的荧光响应随复愈时间上升(图 4A、B)。EC-7与EC-8均具有剂量依赖性, 当诱导剂量为10-1 μg·mL-1时, 两种工程菌的荧光响应效果均达到最佳。在复愈6 h后, EC-7和EC-8在MMC诱导剂量为10-3 μg·mL-1时便可产生显著强于未诱导组的荧光响应, 在MMC诱导剂量为10-1 μg·mL-1时荧光强度达到峰值(图 4C)。在未诱导时, EC-7和EC-8的荧光响应与不含质粒的control组相比, 荧光强度数值达到10倍以上差异, 说明EC-7与EC-8的泄露表达严重。将EC-6、EC-7与EC-8的MMC诱导响应结果进行比较, 3种工程菌的响应趋势基本一致(图 4D)。在复愈时间为6 h时, EC-6与EC-8可达到未诱导组2倍左右的荧光强度, EC-7则只能达到未诱导组1.5倍左右的荧光强度。因此在3种启动回路中选择EC-6与EC-8作为候选工程菌进行下一步比较和筛选。
MMC诱导结果显示目前所构建的传感回路存在泄露表达的问题。泄露表达可能是由于RecA与LexA引起的, 细菌在正常状态下也会存在RecA, 且在转入外源质粒后, 正常细菌中可能并没有足够的LexA与质粒中的SOSBOX相结合, 因此会产生泄露表达。此外, SOSBOX的序列也影响了LexA与其结合的强度, 对泄露表达及响应能力也会产生影响。对工程菌的泄露表达进行表征, 虽然酶标仪无法检测出EC-5诱导前的荧光, 但荧光成像显示EC-5同样存在泄露表达(图 5A)。与EC-7和EC-8相比, EC-6诱导前的荧光信号较弱, 诱导后明显变强, 但强度仍弱于EC-7和EC-8 (图 5B)。EC-7与EC-8诱导前后并无显著差异, 可能是泄露表达所致。荧光成像结果与MMC诱导实验结果趋势一致, EC-6整体荧光强度弱, 对检测手段要求较高, EC-8整体荧光强度强, 但是泄露表达严重, 二者各具优势与不足。
3 候选工程菌的传感性能
用γ射线照射候选工程菌, 比较二者在辐射诱导条件下的传感性能。EC-6和EC-8荧光响应强度随时间逐渐增强, 复愈6 h后EC-6荧光增强趋势减缓, EC-8荧光增强趋势明显减缓(图 6A、B)。在0~60 Gy的辐照中, EC-6具有辐射剂量依赖性, 荧光强度随辐照剂量增强; 在0~50 Gy的辐照中, EC-8具有辐射剂量依赖性, 但当辐照剂量达到60 Gy时, EC-8的荧光响应无明显增加。与未辐照组相比, 在复愈时间6 h后, EC-6与EC-8在辐照剂量为10 Gy时的荧光响应即可具有显著性差异(图 6C)。将两种工程菌的辐射响应结果进行对比, EC-8整体荧光较强, 但泄露表达严重, EC-6响应范围更广, 但荧光强度低(图 6D)。
选择固定诱导剂量, 对EC-6和EC-8在不同时间点的性能进行比较。在10-1 μg·mL-1 MMC和50 Gy辐照诱导后, EC-6与EC-8在复愈1 h时, 诱导组的荧光响应强度均已显著高于未诱导组, 证明两种工程菌响应速度均较快, 辐射响应性能较好(图 7A、B), 但二者用于体内辐射检测的能力仍需要动物模型评估。
4 口服工程益生菌的体内动力学
为确定口服工程菌用于体内辐射检测的相关参数, 首先, 考察工程益生菌在肠道内菌密度的动态变化。构建了组成型表达绿色荧光蛋白的工程菌EC-9, 以便于观察肠道和粪便荧光, 确定后续体内辐照的荧光响应检测时间点。在灌胃2 h后, 大部分荧光仍停留在胃中, 但盲肠以及结直肠中已有荧光, 推测是由于灌胃形式为液体, 大部分菌液可直接通过小肠, 到达盲肠及结直肠, 并沾染在其中的内容物及粪便上, 因此此时盲肠及结直肠中的荧光较强, 但胃中仍有强烈荧光(图 8A)。灌胃48 h后, 肠道中重新出现荧光, 推测是小鼠有进食粪便的习性导致。灌胃2 h后取出的粪便具有强烈荧光, 推测与造成灌胃2 h后肠道荧光分布的原因一致(图 8B)。在灌胃4至8 h间, 粪便中荧光强度成上升趋势, 灌胃8 h后取出的粪便荧光强度达到峰值, 之后逐渐减弱(图 8C)。通过口服工程益生菌的体内动力学表征结果, 确定体内辐照取样时间点为灌胃后8 h。
5 口服工程化微生物传感器体内电离辐射检测性能
根据口服工程益生菌的体内动力学结果确定观测时间点, 小鼠辐照后解剖取肠观察, 可看出ΔEcN未辐照与辐照组的肠道中均无可见荧光, EC-6辐照组的肠道中出现荧光, EC-8未辐照组存在较微弱荧光, 辐照组的肠道中出现荧光, 且EC-8荧光信号响应强度明显强于EC-6 (图 9A)。与体内动力学表征结果相比, 灌胃8 h后, 受照小鼠的胃中及小肠中仍有工程菌残留, 且大多数小鼠的工程菌未到达盲肠与结直肠中, 推测是由于辐照后小鼠肠道受损和辐照前进行麻醉导致工程菌的排出变慢。因此在辐照实验中, 粪便中的荧光检测应晚于8 h。
取灌胃10 h后的小鼠粪便进行荧光检测, ΔEcN为辐照组与辐照组的粪便均无可见荧光, EC-6辐照组的粪便中未出现明显变化, EC-8未辐照组的粪便中存在较弱荧光, 辐照组的粪便中荧光信号强度明显强于未辐照组(图 9B)。对各组粪便样本进行处理后使用酶标仪进行荧光强度的检测, ΔEcN组辐照与未辐照组未表现出显著性差异(图 9C)。EC-6与EC-8辐照组均具有显著高于未辐照组与ΔEcN组的荧光信号响应。EC-6辐照组与未辐照组之间的显著性差异强于EC-8, 推测是误差值导致, 但总体而言, EC-8的整体荧光响应更强, 且能达到未辐照组1.8倍的荧光强度。EC-6辐照组与EC-8未辐照组的粪便在活体成像中显示的荧光较不明显, 但经处理后使用酶标仪检测, 可检测出较高的荧光数值, 推测是细菌大部分被包裹于粪便内部, 表面的活体成像无法检测所导致。出于实际应用与检测的考虑, 结合上述结果, 本研究选用信号响应强, 响应较为明显直观, 便于观察和检测的EC-8作为最终的肠道电离辐射工程菌生物传感器。
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电离辐射作为一种来自于自然界或人为生产的环境因素, 已成为日常生活中的重要工具, 应用于医疗、农业、工业等各个方面。但电离辐射不当使用或失控将导致较大危害, 因此电离辐射的检测一直受到广泛关注。目前电离辐射剂量的检测主要依赖于物理、化学剂量计检测, 无法在生物体内进行辐射检测, 且检测持续性较差, 因此, 迫切需要一种检测手段检测生物体内受到的辐射。
细菌是地球上分布广泛的微生物, 利用生物技术对细菌进行改造, 可以使其识别特定的分析物并表达可用于报告的蛋白质, 构建成为MWCB。MWCB具有强稳定性和环境适应性、成本低、高便捷性的特点, 已在检测重金属、水质毒性等多方面实现了应用[38-40], 在现场检测领域展现出广阔的前景。据报道, 耐辐射奇球菌Deinococcus radiodurans经改造后可在γ射线强度250~2 000 Gy产生绿色荧光, 大肠杆菌经改造后可在γ射线强度0~30 Gy产生绿色荧光, 且二者的荧光强度与电离辐射剂量均呈线性正相关, 显示了微生物对电离辐射生物传感的良好特性[28, 29]。但现有研究中使用的底盘菌安全性较差, 难以应用于生物体内, 检测生物体电离辐射的工程菌有待进一步研究。基于上述背景, 作者利用SOS响应原理及生物体的辐射敏感基因, 选择安全性高的益生菌作为底盘, 成功构建了可体内应用的检测电离辐射的工程菌, 该工程益生菌可持续性检测电离辐射, 可通过粪便中的荧光信号检测实现生物体内的肠道电离辐射检测。
电离辐射可使微生物DNA断裂形成单链DNA(ssDNA), ssDNA可募集RecA和ATP并活化RecA, 活化后的RecA可切割LexA阻遏子。LexA被切割后与SOSBOX分离, 下游基因得以进行表达, 修复DNA。修复完成后, RecA失活, 失去切割LexA的能力, LexA重新与SOSBOX结合并阻遏下游基因表达, 完成SOS响应的整个过程[37]。本研究选择SOS通路中的recA启动子为辐射响应元件, 以荧光蛋白为报告元件构建了辐射响应质粒, 以益生大肠杆菌EcN为底盘菌, 构建了能够辐射剂量依赖性表达荧光蛋白的工程大肠杆菌。为将生物传感器用于体内受照剂量的评估, 分别对底盘菌、基因回路两方面对现有微生物全细胞生物传感器进行优化, 得到了传感性能更优、体内安全性更强的微生物全细胞生物传感器, 并通过动物实验验证了工程益生菌的功能, 现有的工程益生菌传感器可对辐射产生明显荧光响应, 成功实现了生物体内的电离辐射辐射检测。
本研究仍存在不足之处: ①特异性有待提高, 由于电离辐射目前没有特异性强的相关回路, 导致检测电离辐射的工程菌特异性不佳, 多种环境因素都可能会引起基因回路的响应和表达, 需深入研究, 找到电离辐射响应的特异性回路和关键因子; ②泄露表达导致信噪比偏低, 泄露表达问题主要是由于本文中选择的辐射响应回路导致, 解决该问题需要对辐射响应原理进行深入研究, RecA与LexA为SOS响应中的关键调控元件, 通过对二者的表达量进行检测, 根据检测结果, 通过过表达或基因敲除的方式对二者表达量进行调控, 从而降低和抑制泄露表达; ③无法对辐照剂量进行精确定量, 由于存在泄露表达和特异性的问题, 导致目前构建的电离辐射检测工程菌尚未实现辐照剂量的精确定量, 通过寻找特异性更强的传感回路并进行优化, 有望提高辐射检测灵敏度, 实现辐照剂量的定量检测。
工程菌在实际应用中仍存在潜在挑战, 虽然目前EcN作为益生菌, 其安全性已被证实并得到广泛应用, 但改造后的工程益生菌的生物安全性需要进一步进行考证。此外, 目前构建得到的工程菌定植能力较差, 在小鼠肠道中定植时间不足12 h, 后续可通过基因编辑手段改变底盘菌的鞭毛等结构, 增加其定植能力。
本研究以益生菌为底盘, 成功研制了用于体内辐射剂量检测的活体生物药, 安全性高, 对辐射具有明显的荧光响应, 以口服的方式给药, 通过粪便即可实现电离辐射的无创检测, 可用于未来特殊环境作业人群或肿瘤放疗的评估, 为生物体内受照剂量提供了新的检测和评估方法。
作者贡献: 陈紫垣负责实验设计、实验操作、数据分析和文章撰写; 苏云霞、张雅倩、张锋参与实验并进行文章修改; 袁伯川负责课题设计、实验指导、数据分析、文章写作与修改; 金义光负责课题设计、实验指导、数据分析、文章写作与修改。
利益冲突: 本文所有作者声明不存在利益冲突关系。
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2025年第60卷第5期
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doi: 10.16438/j.0513-4870.2025-0208
  • 接收时间：2025-02-27
  • 首发时间：2025-10-29
  • 出版时间：2025-05-12
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  • 收稿日期：2025-02-27
  • 修回日期：2025-04-01
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**袁伯川, E-mail:
金义光, E-mail:
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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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