2023, Vol. 39
http://dx.doi.org/10.13345/j.cjb.220810
中国科学院微生物研究所、中国微生物学会主办
中国科学院微生物研究所、中国微生物学会主办
文章信息
- 张明慧, 张英英, 赵鹏程, 王汉杰
- ZHANG Minghui, ZHANG Yingying, ZHAO Pengcheng, WANG Hanjie
- 基于双工程菌的“锁扣”生物活材料构建及其体内肠道滞留应用
- Construction of "lock-key" biological living material based on double engineered bacteria and its application on intestinal retention in vivo
- 生物工程学报, 2023, 39(3): 1163-1174
- Chinese Journal of Biotechnology, 2023, 39(3): 1163-1174
- 10.13345/j.cjb.220810
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文章历史
- Received: October 8, 2022
- Accepted: January 9, 2023
- Published: January 11, 2023
引用本文
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张明慧, 张英英, 赵鹏程, 王汉杰. 基于双工程菌的“锁扣”生物活材料构建及其体内肠道滞留应用[J]. 生物工程学报, 2023, 39(3): 1163-1174.
ZHANG Minghui, ZHANG Yingying, ZHAO Pengcheng, WANG Hanjie. Construction of "lock-key" biological living material based on double engineered bacteria and its application on intestinal retention in vivo[J]. Chinese Journal of Biotechnology, 2023, 39(3): 1163-1174.
基于双工程菌的“锁扣”生物活材料构建及其体内肠道滞留应用
1. 天津大学生命科学学院, 天津 300072;
2. 徐州医科大学医学影像学院, 江苏 徐州 221004
2. 徐州医科大学医学影像学院, 江苏 徐州 221004
摘要：生物活材料的研究主要集中在利用单一细菌生产生物膜、水塑料等体外应用。由于菌株尺寸较小，当其应用于体内时，容易发生逃逸，导致滞留效果较差。为解决这一难题，本研究借助大肠杆菌(Escherichia coli)表面展示系统(Neae)，在两个菌株表面分别展示SpyTag和SpyCatcher，构建一种双菌“锁扣”型生物活材料生产系统。两菌株之间通过SpyTag和SpyCatcher的结合，发生原位交联，从而长时间滞留在肠道部位。体外实验表明两菌株混合几分钟后，会发生明显的沉降。此外，利用共聚焦成像和微流控平台进一步证明了该系统在流动状态下的粘附效果。最后，为了验证该系统在体内应用的可行性，小鼠连续3 d口服A菌(p15A-Neae-SpyTag/sfGFP)和B菌(p15A-Neae-SpyCatcher/mCherry)，收集肠道组织进行冷冻切片染色。结果表明，相较于未结合菌株，该双菌系统能更多滞留在小鼠肠道，为生物活材料进一步的体内应用奠定了基础。
关键词：生物活材料 表面展示 SpyTag与SpyCatcher 双菌
Construction of "lock-key" biological living material based on double engineered bacteria and its application on intestinal retention in vivo
1. School of Life Sciences, Tianjin University, Tianjin 300072, China;
2. School of Medical Imaging, Xuzhou Medical University, Xuzhou 221004, Jiangsu, China
2. School of Medical Imaging, Xuzhou Medical University, Xuzhou 221004, Jiangsu, China
Abstract: At present, the research of biological living materials mainly focuses on applications in vitro, such as using a single bacterial strain to produce biofilm and water plastics. However, due to the small volume of a single strain, it is easy to escape when used in vivo, resulting in poor retention. In order to solve this problem, this study used the surface display system (Neae) of Escherichia coli to display SpyTag and SpyCatcher on the surface of two strains, respectively, and constructed a double bacteria "lock-key" type biological living material production system. Through this force, the two strains are cross-linked in situ to form a grid-like aggregate, which can stay in the intestinal tract for a longer time. The in vitro experiment results showed that the two strains would deposit after mixing for several minutes. In addition, confocal imaging and microfluidic platform results further proved the adhesion effect of the dual bacteria system in the flow state. Finally, in order to verify the feasibility of the dual bacteria system in vivo, mice were orally administrated by bacteria A (p15A-Neae-SpyTag/sfGFP) and bacteria B (p15A-Neae-SpyCatcher/mCherry) for three consecutive days, and then intestinal tissues were collected for frozen section staining. The in vivo results showed that the two bacteria system could be more detained in the intestinal tract of mice compared with the non-combined strains, which laid a foundation for further application of biological living materials in vivo.
Keywords:
biological living materials surface display SpyTag-SpyCatcher bi-bacteria
传统的生物材料，如无机纳米材料、碳纳米材料、高分子纳米材料以及超分子纳米材料等[1]，在骨修复[2-3]、伤口愈合[4-6]、肿瘤诊治疗[7-9]、成像[8]以及脑疾病治疗[10]等方面应用广泛。但是，其合成过程繁琐，物理结构、表面性质和固有功能难以改变，并且它们在体内应用过程中也出现了无法自主调节、持续时间短等问题。因此，需要开发一种可编程、能够自我调节的新型生物活材料[11]来满足体内应用的各种需求。
近几年，新型生物活材料的研究获得进展，其中生物被膜的研究最为广泛[12-15]。例如，哈佛大学Neel S. Joshi教授团队利用生物被膜开发了一种可降解“水塑料”[16]，用于解决日益严重的塑料污染问题。此外，借助环境响应型基因表达开关，科学家们开发了可穿戴生物传感器，从而实现无创、实时健康监测[17-19]。美国马萨诸塞大学研究团队将生物被膜设计成皮肤贴片，通过感知汗液，为可穿戴设备发电[20]。深圳先进技术研究院钟超课题组利用大肠杆菌和枯草芽孢杆菌生物被膜的CsgA蛋白来生产用于水下粘合的活体胶水[21-23]；此外，在CsgA蛋白表面融合其他功能蛋白，研发用于环境消毒[24]、黏膜修复[25-26]以及生物催化[27]的新型生物活材料。
目前，新型生物活材料的研究集中在利用单一菌株的生物被膜来生产各种功能活材料，并探究其体外应用。研究发现[28]：当单一菌株应用到体内时，其难以在靶部位长时间滞留，容易发生逃逸，导致治疗效果大打折扣。随着合成生物学技术的发展，生物活材料也逐步应用到体内。为了使其更好地在体内发挥作用，需要开发一种能够长时间滞留在体内的生物活材料[29-30]。深圳先进技术研究院戴卓君课题组利用大肠杆菌反向自动转运蛋白和表面展示系统[31-32]，将抗原、抗体分别展示在两个菌株表面，构建一种可原位交联的生物活材料，并将其用于3D打印、有机磷农药降解、海藻糖合成和可穿戴生物传感器。考虑到肠道环境的复杂性，本研究采用SpyTag和SpyCatcher这一更加稳定共价连接系统[33]，将其分别展示在两菌株表面[34]，用于开发一种新型可编程、能在肠道持久滞留的生物活材料。这一活材料在体内外可以稳定地交联成网格状结构，从而使菌株更多、更持久滞留在肠道部位。我们相信这种新型生物活材料为肠道疾病、肿瘤等重大疾病的高效治疗提供新思路。
1 材料与方法 1.1 材料 1.1.1 菌株和质粒本文所用的菌株和质粒详见表 1。
表 1 本文所用菌株和质粒
Table 1 Strains and plasmids used in this study
| Strain and plasmid | Feature | Source |
| E. coli DH5α | Cloned strain | In the lab |
| E. coli BL21 | Functional verification | In the lab |
| pRSF-J23100-sfGFP | Green fluorescent protein tag | In the lab |
| mCherry | Red fluorescent protein tag | In the lab |
| p15A-Neae-SpyTag | SpyTag displayed on the bacteria surface | Constructed in this study |
| p15A-Neae-SpyCatcher | SpyCatcher displayed on the bacteria surface | Constructed in this study |
| p15A-Neae-SpyTag-SnoopTag | SpyTag and SnoopTag displayed on the surface | Constructed in this study |
| pUC19-SnoopCatcher | Expressed SnoopCatcher | Constructed in this study |
本文所用引物如表 2所示。
表 2 本研究所用引物
Table 2 PCR primes used in this study
| Primer name | Primer sequence (5′→3′) |
| p15a-gj-tag-f | ATGCATACAAGCCGACGAAGTAATAAAGCCAGGCATCAAATAAAACGAAAGGCTCA |
| p15a-gj-tag-r | ATATCATGCGGAATATTCAGAGAAAGAATATCCTGCTTCTTGTACTCCAG |
| p15a-pd-tag-f | CTGAATATTCCGCATGATATTAATGGTACTGAACAC |
| p15a-pd-tag-r | CTTCGTCGGCTTGTATGCATCAACCATGACAATATGGGCTCTAGTCGCACCATCAAAAAATATAACCGCA |
| p15a-gj-SpyCatcher-F | AAGGTGACGCTCATATTGATTAATAAAGCCAGGCATCAAATAAAACGAAAGG |
| p15a-gj-SpyCatcher-R | GATAAGGTATCAACCATGGCTCTAGTCGCACCATCAAAAAATATAACCGCA |
| SpyCatcher-F | GCCATGGTTGATACCTTATCAGGTTTATCAAG |
| YZ-SpyCatcher-R | ATCAATATGAGCGTCACCTTTAGTTGCTT |
| snoop-spy-gj-F | TTATTATCACTTATTCAGGCGTAGCACCAGGCGTTTAAGGG |
| snoop-spy-gj-R | TCGCCCAGTTTTCCCATATGACCAGATCCGCCCTTCGTCGGCTTGTATGCATCAAC |
| SnoopTag-PD-F | CATATGGGAAAACTGGGCGATATTGAATTTATTAAAGTGAACAAATAATAAAGCCAGGCATCAAATAAAACGAAAGG |
| JP-R-3Tag | GCCTGAATAAGTGATAATAAGCGGATGAATG |
| SC-GJ-F | TAAAGCGCTGCATGCCTAGTG |
| SC-GJ-R | CTCTAAACAAAATTATTCATTATACGAGCCGGA |
| SC-PD-F-2 | ATGAATAATTTTGTTTAGAGAAAGAGGAGAAATACTAGATGCATATGAAGC |
| SC-PD-R-2 | ACTAGGCATGCAGCGCTTTAATGATGGTGATGGTGATGTTTCGG |
胰蛋白胨、酵母提取物购自OXOID公司；氯化钠和琼脂粉购自北京索莱宝科技有限公司；本文所有引物和Neae表面展示基因由金唯智公司合成；氯霉素、硫酸卡那霉素和氨苄青霉素粉末购自北京索莱宝科技有限公司; Prime STAR Max Premix (2×)购自TaKaRa公司；重组酶、2×Rapid Taq Master Mix、质粒小提试剂盒、纯化试剂盒、DNA Marker和10×DNA上样缓冲液购自南京诺唯赞生物科技股份有限公司；恒温培养摇床购自天津欧诺仪器股份有限公司；电泳仪和PCR仪购自伯乐公司；微型注射泵购自深圳市瑞沃德生命科技有限公司；多功能酶标仪购自赛默飞世尔科技公司；共聚焦显微镜购自尼康公司。
1.2 方法 1.2.1 质粒构建质粒构建按参考文献中所述方法[35]。
1.2.2 两菌株粘附的验证将测序正确的p15A-Neae-SpyTag/sfGFP和p15A-Neae-SpyCatcher/mCherry质粒分别转化到大肠杆菌BL21中，构建A菌(p15A-Neae-SpyTag/sfGFP)和B菌(p15A-Neae-SpyCatcher/mCherry)。随后接种到含氯霉素和氨苄青霉素的液体LB培养基，37 ℃过夜培养。第2天，按照1:100的比例将其接种到15 mL LB培养基中，继续培养24 h。将两菌株等量混合后静置，在不同时间点，收集菌液上清，用多功能酶标仪测定上清OD600值的变化。
分别取2 mL A菌和B菌，在透明的玻璃管中等量混合后静置，在0、10、60 min这3个时间点进行拍照。
1.2.3 激光共聚焦显微镜观察两菌株粘附在验证菌株间的交联时，我们设置了4组实验，分别是对照组(sfGFP+mCherry、A菌+ mCherry、sfGFP+B菌)和实验组(A菌+B菌)。对于实验组，分别取10 μL的A菌和B菌，先后滴在同一载玻片的相同位置，静置片刻，将盖玻片覆盖在载玻片上。对照组操作同上。利用激光共聚焦显微镜来观察绿色荧光和红色荧光的重叠效果。
1.2.4 p15A-Neae-SpyTag-SnoopTag和p15A-Neae-SpyCatcher粘附的验证为了进一步验证两菌株的交联，将p15A-Neae-SpyTag-SnoopTag和p15A-Neae-SpyCatcher质粒分别转化到BL21菌株中。按此前同类方法培养，混合静置后，测定上清OD600的变化。
1.2.5 SnoopCatcher蛋白的纯化将测序正确的pUC19-SnoopCather质粒转化到BL21菌株中，37 ℃过夜培养。第2天，将其接种到1 L的LB培养基中，37 ℃培养24 h。4 000 r/min离心20 min，收集菌体沉淀。加入裂解液(20 mmol/L Tris, 300 mmol/L NaCl, pH 8.0)重悬菌体，超声破碎40 min。破碎完成后，18 000 r/min离心30 min，收集上清。通过0.22 μm滤膜过滤、镍柱纯化，用超滤管对蛋白进行浓缩。
1.2.6 采用微流控芯片技术来验证两菌株的粘附微流控芯片按文献[36]所述来制作。芯片尺寸为1 500 mm×500 mm，深度是100 μm。聚二甲基硅氧烷(polydimethylsiloxane, PDMS)层与玻璃片键合之前，先对玻璃片进行活化。活化的玻璃片经烷化处理，被修饰上氨基。通过氨基-羧基之间的共价结合反应，SnoopCatcher蛋白成功结合到微流控芯片上。A菌以40 μL/min流速流通3 h。随后，将去离子水以20 μL/min的流速来冲洗掉未结合A菌。最后，表达mCherry的菌株和B菌分别以40 μL/min流速流通3 h。利用去离子水冲洗未结合的mCherry菌株和B菌株，并将其置于激光共聚焦显微镜下观察。
1.2.7 体内粘附效果的验证将雌性BALB/c小鼠随机分成对照组(sfGFP+mCherry)和实验组(A菌+B菌)，n=3。对照组小鼠灌胃200 μL过夜培养的sfGFP菌株，实验组小鼠灌胃200 μL过夜培养的A菌。1 h后，对照组小鼠灌胃同等体积的mCherry菌株，实验组小鼠灌胃同等体积的B菌。连续灌胃3 d，第4天不做任何处理，第5天重复此前操作。4 h后，收集小鼠结肠、空肠、回肠、十二指肠段进行冷冻切片，经Hoechst染色后，置于激光共聚焦显微镜下观察。本文动物实验方案遵守相关实验动物福利规定，并获得中国医学科学院放射医学研究所实验动物伦理委员会批准，批准号为SYXK (津)：2019-0002。
2 结果与分析 2.1 A菌(p15A-Neae-SpyTag/sfGFP)和B菌(p15A-Neae-SpyCatcher/mCherry)粘附的验证肠出血型大肠杆菌的内膜N端，是一个反向自动转运蛋白和表面展示系统。基于此，SpyTag和SpyCatcher可展示在两菌株表面(图 1)。将两菌株等量混合，静置。结果显示：静置10 min时，约25%菌株粘附并沉降下来。静置60 min后，60%以上菌株明显聚集并沉降(图 2、图 3)。而表面未展示SpyTag和SpyCatcher的菌株，在所监测的3 h内并未发生任何聚集现象(图 3)。
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| 图 1 A菌(左)和B菌(右)质粒构建示意图 Fig. 1 The schematic diagram of plasmids of bacteria A (left) and B (right). |
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| 图 2 菌株等量混合的宏观沉降图 Fig. 2 Macro sedimentation diagram of equal mixing of strains. (A): Macro-sedimentation diagram of strains separately expressing sfGFP and mCherry mixing in equal amounts. G: sfGFP; R: mCherry; M: sfGFP+mCherry. (B): Macro-sedimentation diagram of macro sedimentation diagram of mixed bacteria A and B in equal amount. A: p15A-Neae-SpyTag/sfGFP; B: p15A-Neae-SpyCatcher/mCherry; Mix: p15A-Neae-SpyTag/sfGFP+p15A-Neae-SpyCatcher/mCherry. (A)：单独表达sfGFP和mCherry的菌株等量混合后的宏观沉降图. G：sfGFP；R：mCherry；M：sfGFP+mCherry. (B)：A菌和B菌等量混合后的宏观沉降图. A：p15A-Neae-SpyTag/sfGFP；B：p15A-Neae-SpyCatcher/mCherry；Mix：p15A-Neae-SpyTag/sfGFP+p15A-Neae-SpyCatcher/mCherry |
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| 图 3 A菌和B菌粘附的定量验证 Fig. 3 Quantitative verification of adherence of bacteria A and B. |
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当该双菌系统应用于体内时，双菌数量无法严格控制。因此，我们探究了A菌与B菌在1:0.25、1:0.50、1:0.75比例下的粘附情况。结果表明：随着B菌数量的增加，A菌和B菌粘附速率增加(图 4A)。为了增加该双菌系统的功能性，我们引入了一对与SpyTag-SpyCatcher正交的共价偶联系统(SnoopTag-SnoopCatcher)，在p15A-Neae-SpyTag质粒上延伸出一个SnoopTag位点。随后探讨了SnoopTag的加入是否会影响粘附效果。结果表明，在最初30 min内，p15A-Neae-SpyTag-SnoopTag菌株与B菌粘附效果不明显。随着时间延长，该菌株和B菌的粘附与A、B两菌的粘附效果类似(图 4B)。最后，我们又尝试了3个串联的SpyTag展示在A菌表面，探究其与B菌的粘附效果。结果表明：串联3个SpyTag不仅影响两菌株粘附速度，粘附效果也大大降低(图 4C)。这可能是由于延伸出的链过长，阻碍了邻近位点结合。
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| 图 4 上清液中剩余的细菌含量 Fig. 4 Residual bacterial content in the supernatant. A: The content change of residual bacteria in supernatant after mixing bacteria A and B in different proportions. B: The content change of residual bacteria in supernatant after mixing p15A-Neae-SpyTag-SnoopTag strains and bacteria B for different times. C: The content change of residual bacteria in supernatant after mixing p15A-Neae-3SpyTag strains and bacteria B for different times. A：不同比例A菌和B菌混合后，上清中剩余细菌的含量变化. B：p15A-Neae-SpyTag-SnoopTag菌株与B菌混合不同时间后，上清中剩余细菌的含量变化. C：p15A-Neae-3SpyTag菌株与B菌混合不同时间后，上清中剩余细菌的含量变化 |
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除了从宏观角度观察两菌株的粘附外，我们还从微观角度进行了验证。分别将对照组(sfGFP+mCherry、A菌+mCherry、sfGFP+B菌)和实验组(A菌+B菌)的两菌株混合，并置于低倍镜下观察。如图 5A所示，与对照组相比，实验组绿色荧光和红色荧光重叠效果较好。将其置于100倍油镜下观察，可以更清楚地看到A菌和B菌的粘附现象(图 5B)。
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| 图 5 激光共聚焦显微镜观察菌株的粘附现象 Fig. 5 Observation of bacterial adhesion by laser confocal microscopy. A: The adhesion effects of sfGFP+mCherry, bacteria A+mCherry, sfGFP+bacteria B, and bacteria A+bacteria B were observed under a laser confocal microscope (10×). B: The adhesion effects of sfGFP+mCherry, bacteria A+mCherry, sfGFP+bacteria B, and bacteria A+bacteria B were observed under a laser confocal microscope (100×). A：在激光共聚焦显微镜10倍镜下观察sfGFP+mCherry、A菌+mCherry、sfGFP+B菌、A菌+B菌的粘附. B：在激光共聚焦显微镜100倍镜下观察sfGFP+mCherry、A菌+mCherry、sfGFP+B菌、A菌+B菌的粘附 |
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利用微流控平台对p15A-Neae-SpyTag-SnoopTag菌株与B菌的粘附进行了验证。首先将SnoopCatcher蛋白固定在微流控芯片上，通过SnoopTag-SnoopCatcher之间的共价结合，将p15A-Neae-SpyTag-SnoopTag菌株固定在微流控芯片的指定通道，然后流通B菌(图 6A)。与未展示SpyCatcher的菌株相比，表面展示SpyCatcher的菌株能够更好地与p15A-Neae-SpyTag-SnoopTag菌株结合(图 6B)，从而发生粘附。
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| 图 6 流动状态下两菌株的粘附 Fig. 6 Adhesion of two strains in flowing state. A: The schematic diagram of microfluidic controlling process. (1) SnoopCatcher protein was fixed on the chip; (2) p15A-Neae-SpyTag-SnoopTag strains were injected; (3) B strains were combined with the previous strain. B: The adhesion of p15A-Neae-SpyTag-SnoopTag strains and bacteria B observed by a laser confocal microscope. A：微流控操作过程示意图. (1) 先将SnoopCatcher蛋白固定在芯片上；(2) 然后流通p15A-Neae-SpyTag-SnoopTag菌株；(3) 最后流通B菌，使其与p15A-Neae-SpyTag-SnoopTag结合. B：显微镜观察p15A-Neae-SpyTag-SnoopTag菌株和B菌的粘附情况 |
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为了验证“锁扣”型生物活材料在体内应用的效果，连续3 d先后给小鼠口服A菌和B菌，在第5天收集结肠、空肠、回肠、十二指肠进行冷冻切片，然后置于共聚焦显微镜下观察。结果显示(图 7A)：与单独口服sfGFP或mCherry菌株相比，A菌和B菌在结肠、空肠和十二指肠的粘附效果更加明显。在回肠部位，两者区别不大。此外，荧光定量分析结果(图 7B)与图 7A一致。以上结果表明，该双菌体系在肠道内可以很好地粘附，并且滞留效果也要优于单一菌株，有效提高了工程菌在肠道的滞留效率，有望在体内得到广泛的应用。
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| 图 7 “锁扣”型生物活材料在肠道内的滞留 Fig. 7 Retention of "lock-key" biological living materials in intestine. A: Left is the adhesion of bacteria A and B in different intestinal segments of mice (colon, ileum, jejunum, and duodenum). Right is the adhesion of individual sfGFP strains and mCherry strains in different intestinal segments. B: Quantitative analysis of fluorescence intensity in Figure 7A. Left: Quantitative analysis of Hoechst fluorescence intensity; Intermediate: Quantitative analysis of sfGFP fluorescence intensity; Right: Quantitative analysis of mCherry fluorescence intensity. A：左边为A菌和B菌在小鼠不同肠段(结肠、回肠、空肠和十二指肠)的粘附情况；右边为单独的sfGFP菌株和mCherry菌株在不同肠段的粘附情况. B：图 7A的荧光强度定量分析(左边：Hoechst荧光强度定量分析；中间：sfGFP荧光强度定量分析；右边：mCherry荧光强度定量分析) |
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生物活材料的可编程性、持续性以及动态可调性，在其生物医学应用方面具有重要意义。相较于传统的生物材料，生物活材料不仅可以精准到达病灶，减少毒副作用，还可以通过在病灶部位的定植，持久发挥作用。而且，合成生物学技术可以给生物活材料定制不同功能。因此，生物活材料在生物催化、皮肤修复、环境污染治理、仿生器官[37]以及某些疾病诊疗等方面广泛应用。
截至目前，生物活材料多集中在体外应用。单一菌株在体内发挥作用时，滞留效率低，限制了进一步应用。本研究对微生物进行基因工程改造，利用菌株间的交联形成大的网格状聚集体，能够帮助菌株长久滞留在体内。在后续研究中，可以引入治疗物质，从而拓宽生物活材料的应用范围。本研究可以为新型生物活材料的体内应用提供新的思路。
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