两株CRISPR工程大肠埃希菌在小鼠体内的定植实验研究

doi:10.12300/j.issn.1674-5817.2022.088

实验动物与比较医学 ›› 2022, Vol. 42 ›› Issue (6): 541-550.DOI: 10.12300/j.issn.1674-5817.2022.088

两株CRISPR工程大肠埃希菌在小鼠体内的定植实验研究

任晨吟¹, 高思琦¹, 李浩², 唐标³, 杨华³, 刘月环⁴()()

^1.杭州医学院临床医学院, 杭州 310053
^2.杭州医学院护理学院, 杭州 310053
^3.浙江省农业科学院农产品质量安全与营养研究所, 杭州 310021
^4.杭州医学院浙江省实验动物与安全性研究重点实验室, 杭州 310013

收稿日期:2022-06-17 修回日期:2022-08-31 出版日期:2022-12-25 发布日期:2023-01-04
通讯作者: 刘月环（1974—），女，医学博士，研究员，研究方向：微生物组基因编辑与耐药性防控技术的研究。E-mail： 215193122@qq.com。ORCID： 0000-0003-3961-1362
作者简介:任晨吟（2001—），女，本科在读，研究方向：微生物耐药机制。E-mail：2490394878@qq.com
基金资助:
浙江省重点研发计划项目“CRISPR工程菌消减动物源耐药基因研究”(2020C02031)

In Vivo Colonization Test of Two CRISPR-Engineered Escherichiacoli in Mice

Chenyin REN¹, Siqi GAO¹, Hao LI², Biao TANG³, Hua YANG³, Yuehuan LIU⁴()()

^1.School of Clinical Medicine, Hangzhou Medical College, Hangzhou 310053, China
^2.School of Nursing, Hangzhou Medical College, Hangzhou 310053, China
^3.Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
^4.Zhejiang Key Laboratory of Experimental Animals and Safety Research, Hangzhou Medical College, Hangzhou 310013, China

Received:2022-06-17 Revised:2022-08-31 Published:2022-12-25 Online:2023-01-04
Contact: LIU Yuehuan (ORCID: 0000-0003-3961-1362), E-mail: 215193122@qq.com

摘要/Abstract

摘要：

目的利用小鼠对2株大肠埃希菌工程菌Nissle1917和BW25113在肠道的定植能力与定植效率进行评价，以筛选出肠道定植效率高的菌株，为后续利用成簇的规律性间隔的短回文重复序列（clustered regularly interspersed short palindromic repeats，CRISPR）及其相关蛋白（CRISPR associated protein，Cas）系统的工程菌消减体内耐药菌的研究奠定基础。方法每株工程菌的实验共取70只18～20 g的ICR小鼠，雌雄各半，操作时小鼠区分性别并随机分为7个处理组，每组10只（6只实验，4只对照）。实验组灌胃2×10 ¹⁰个工程菌共200 μL，对照组灌胃等体积的PBS。灌胃后1、3、6、12、24、48、72 h分别取小鼠的肠系膜淋巴结、胃、回盲部、结肠组织及内容物，分别采用平板划线、荧光镜检和PCR法检测工程菌，比较Nissle1917和BW25113这2菌株在小鼠体内的定植能力与效率。结果灌胃后前6个时段（1、3、6、12、24、48 h）中，3种方法检测到2株菌在胃、回盲部、结肠组织有定植，淋巴结未检到目标菌株；而72 h有且仅有Nissle1917菌定植于回盲部和结肠组织，Nissle1917和BW25113两株菌的定植效率分别为100%和0。结论 Nissle1917菌定植效率高于BW25113菌，且能较长时间定植于回盲部和结肠组织，提示其可作为防控耐药基因传播用CRISPR系统的备选载体菌。

关键词: CRISPR工程大肠埃希菌, 肠道定植能力, 定植效率, ICR小鼠

Abstract:

Objective The colonization ability and efficiency of two Escherichia coli ( E. coli)-engineered strains, Nissle1917 and BW25113, in the intestine were evaluated in mice, we aimed to screen out strains for subsequent research on clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system-engineered bacteria to eliminate drug-resistant bacteria via high intestinal colonization efficiency. Methods Seventy ICR mice (18–20 g), half male and half female, were randomly divided into 7 treatment groups by gender, with 10 mice in each group (6 for experiment and 4 for control). The experimental group was gavaged with 2×10 ¹⁰ of the engineered strains at a final volume of 200 μL, and the control group was gavaged with an equal volume of PBS. At 1, 3, 6, 12, 24, 48, and 72 hours after gavage, the mesenteric lymph nodes, stomach, ileocecal and colonic tissues, and intestinal contents of the mice were removed. The two E. coli strains were detected using plate inoculation, fluorescence microscopy, and PCR amplification to compare their in vivo colonization ability and efficiency. Results At 1, 3, 6, 12, 24, and 48 hours after gavage, both E. coli strains had colonized in the stomach, ileocecal and colonic tissues as detected using the three methods, and no leakage of E. coli fluid from the lymph nodes was observed; at 72 hours, only Nissle1917 colonized in the ileocecal and colonic tissues, comparing the colonization efficiency of the two E. coli strains, that of Nissle1917 was 100% and that of BW25113 was 0 at 72 hours. Conclusion Nissle1917 has a higher colonization efficiency than BW25113 and can colonize in the mucosal surface of ileocecal and colonic tissues for a long time, suggesting that it can be used as a carrier for the CRISPR system to prevent and control drug resistance gene transmission.

Key words: CRISPR engineered Escherichia coli, Intestinal colonization capacity, Colonization efficiency, ICR mouse

中图分类号:

Q95-33

任晨吟, 高思琦, 李浩, 唐标, 杨华, 刘月环. 两株CRISPR工程大肠埃希菌在小鼠体内的定植实验研究[J]. 实验动物与比较医学, 2022, 42(6): 541-550.

Chenyin REN, Siqi GAO, Hao LI, Biao TANG, Hua YANG, Yuehuan LIU. In Vivo Colonization Test of Two CRISPR-Engineered Escherichiacoli in Mice[J]. Laboratory Animal and Comparative Medicine, 2022, 42(6): 541-550.

图/表 6

图1 大肠埃希菌I型CRISPR-Cas系统结构示意（A）以及Nissle1917菌株中pGLO-J23100-GFP质粒图谱（B）和BW25113菌株中pGLO-araC-GFP质粒图谱（C）注：J23100，组成型启动子；RBS，核糖体结合位点；GFP，绿色荧光蛋白表达基因（荧光报告系统元件）；Promoter，基因启动子；Amp R，氨苄青霉素抗性筛选标记基因；araC，ara操纵子调节蛋白。

Figure 1 Structure of the E. coli I CRISPR-Cas system (A), plasmid profile of pGLO-J23100-GFP in Nissle1917 (B), and plasmid profile of pGLO-araC-GFP in BW25113 (C)Note：J23100， constitutive promoter； RBS， ribosome binding site； GFP， green fluorescent protein expression gene （fluorescent reporter system element）； Promoter， gene promoter； Amp R， ampicillin resistance selection marker gene； araC， ara operon regulatory protein.

图2 工程菌原液和稀释液以及饲料和10%葡萄糖水的荧光检测结果注：A，工程菌原液（×100）；B~C，工程菌稀释液（B，×100；C，×1 000）；D~F，饲料（D，绿荧光；E，红荧光；F，蓝荧光）（×200）；G，10%葡萄糖水（×200）。

Figure 2 Fluorescence in stock solution and dilution solution of E. coli engineered bacteria as well the diet and 10% glucose waterNote: A, Stock solution of engineered bacteria (100×); B-C, Dilution solution of engineered bacteria (B,×100; C, ×1 000); D-F, Green, red and blue fluorescence in diet (×200); G, Fluorescence in 10% glucose water (×200)

图3 Amp +平板检测2株工程菌在小鼠体内各部位的定植情况注：A~C依次为灌胃后24 h、48 h、72 h的Nissle1917菌；D~F分别为灌胃后24 h、48 h、72 h的BW25113菌；1、2、3为工程菌实验组，4、5为PBS对照组；同一平板左上、右上、左下、右下依次为肠系膜淋巴结、胃、回盲部、结肠。

Figure 3 Amp + plate detection of the colonization of two E. coli strains in mouse tissuesNote: A-C, Nissle1917 at 24, 48, and 72 h after gavage, respectively；D-F, BW25113 at 24, 48, and 72 h after gavage, respectively; 1, 2, and 3 are engineered E. coli experimental groups; 4 and 5 are the PBS control groups; from the left to the right, and from the top to the bottom on the same plate: mesenteric lymph nodes, stomach, ileocecal and colon tissues.

图4 荧光镜检法检测灌胃后24 h、48 h和72 h小鼠肠道组织中2株工程菌的黏附情况（×200）注：A，灌胃后24 h回盲部Nissle1917；B， 24 h结肠Nissle1917；C， 24 h回盲部BW25113；D， 24 h结肠BW25113；E， 48 h回盲部Nissle1917；F， 48 h结肠Nissle1917；G， 48 h回盲部BW25113；H， 48 h结肠BW25113；I， 72 h回盲部Nissle1917；J， 72 h结肠Nissle1917；K， 72 h 回盲部BW25113；L， 72 h结肠BW25113。箭头示黏附的发光的细菌。

Figure 4 Fluorescence microscopic detection of adhesion of 2 strains of engineered E. coli in the intestinal tissue of mice at 24 h, 48 h, and 72 h after gavage (×200)Note: A, Nissle1917 in the ileocecal area at 24 h; B, Nissle1917 in the colon at 24 h; C, BW25113 in the ileocecal area at 24 h; D, BW25113 in the colon at 24 h; E, Nissle1917 in the ileocecal area at 48 h; F, Nissle1917 in the colon at 48 h; G, BW25113 in the ileocecal area at 48 h; H, BW25113 in the colon at 48 h; I, Nissle1917 in the ileocecal area at 72 h; J, Nissle1917 in the colon at 72 h; K, BW25113 in the ileocecal area at 72 h; L, BW25113 in the colon at 72 h. Arrows show attached glowing bacteria.

图5 工程菌Nissle1917和BW25113的PCR产物电泳注：M，DNA marker（DL2 000）；P，阳性对照（Nissle1917/BW25113）；N，阴性对照（PBS）；1-6泳道，灌胃24 h（1、3、5泳道为回盲部，2、4、6泳道为结肠）；7-12泳道，灌胃48 h（7、9、11泳道为回盲部，8、10、12泳道为结肠）；13-18泳道，灌胃72 h（13、15、17泳道为回盲部，14、16、18泳道为结肠）。

Figure 5 Electrophoresis of PCR products of Nissle1917 and BW25113Note： M, DNA marker (DL2 000); P, Positive control (Nissle1917/BW25113); N, Negative control (PBS). Lanes 1–6: Gavage 24 h (lanes 1, 3 and 5 show ileocecal, lanes 2, 4 and 6 show colon); Lane 7-12: Gavage 48 h (lanes 7, 9 and 11 show ileocecal, lanes 8, 10 and 12 show colon); Lane 13-18: Gavage 72 h (lanes 13, 15 and 17 show ileocecal, lanes 14, 16 and 18 show colon).

图6 工程菌Nissle1917和BW25113灌胃24～72 h后在小鼠不同组织中的定植效率比较

Figure 6 Comparison of the colonization efficiency of Nissle1917 and BW25113 (gavage 24–72 h) in different tissues of mice

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两株CRISPR工程大肠埃希菌在小鼠体内的定植实验研究

In Vivo Colonization Test of Two CRISPR-Engineered Escherichiacoli in Mice

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