大肠杆菌DH5α通过外膜囊泡传递抗生素抗性基因AmpR

doi:10.13560/j.cnki.biotech.bull.1985.2024-0335

摘要/Abstract

摘要：

【目的】 探究大肠杆菌（Escherichia coli）DH5α 外膜囊泡（outer membrane vesicles, OMVs）在细菌间传递抗生素抗性基因AmpR的能力。【方法】通过超速离心法提取外膜囊泡；然后通过透射电镜，纳米颗粒跟踪分析（NTA）以及Western blot检测和表征OMVs的形态、粒径和浓度。同时 PCR鉴定OMVs中的氨苄青霉素抗性基因AmpR。将OMVs与大肠杆菌DH5α共孵育培养，观察细菌在氨苄抗生素培养基上的生长情况，挑取单菌落进行菌落PCR鉴定AmpR的存在。【结果】所提取的囊泡的平均粒径约为125 nm，在OMVs粒径范围之内，OMVs标志蛋白OmpC阳性，显示所提取的囊泡为细菌外膜囊泡OMVs。同时PCR鉴定该OMVs中含有氨苄青霉素抗性基因AmpR。与OMVs共孵育培养后的大肠杆菌DH5α能在LB固体平板上生长，通过菌落PCR鉴定大肠杆菌中含有AmpR基因。【结论】具有抗性基因的大肠杆菌Escherichia coli DH5α可以通过外膜囊泡OMVs将AmpR传递给周围的大肠杆菌，实现耐药性转移。

关键词: 大肠杆菌, 外膜囊泡, 氨苄抗性基因, 基因转移, 耐药性

Abstract:

【Objective】 To investigate the ability of Escherichia coli DH5α outer membrane vesicles（OMVs）transferring the antibiotic resistance gene AmpR among bacteria. 【Method】 The OMVs were extracted by ultracentrifugation; then the morphology, particle size and concentration of OMVs were detected and characterised by transmission electron microscopy, nanoparticle tracking analysis（NTA）and Western blot. Meanwhile, PCR was performed to identify AmpR, an ampicillin resistance gene in OMVs. OMVs were co-incubated and cultured with blank Escherichia coli DH5α, and the bacterial growth on medium with ampicillin was observed, and single colonies were picked to analysize the presence of AmpR by PCR. 【Result】 The average particle size of the extracted vesicles was 125 nm, which was within the range of OMVs particle size, and the OMVs marker protein OmpC was positive, indicating that the extracted vesicles were bacterial OMVs. It was also identified that the ampicillin resistance gene AmpR was in these OMVs. After co-incubation with OMVs, blank E. coli DH5α grew on LB solid plates, and AmpR gene was identified by colony PCR in these bacterial cells. 【Conclusion】 Escherichia coli DH5α with resistant genes can transfer resistance by passing AmpR to surrounding E. coli via outer membrane vesicles OMVs.

Key words: Escherichia coli, outer membrane vesicles（OMVs）, genes with ampicillin resistanc, gene transfer, resistance to drug

庄棵, 梁至轩, 何英婷, 谢秋玲. 大肠杆菌DH5α通过外膜囊泡传递抗生素抗性基因AmpR[J]. 生物技术通报, 2024, 40(12): 275-281.

ZHUANG Ke, LIANG Zhi-xuan, HE Ying-ting, XIE Qiu-ling. Transfer of Antibiotic-resistance Gene AmpR by Escherichia coli DH5α Through Outer Membrane Vesicles[J]. Biotechnology Bulletin, 2024, 40(12): 275-281.

图/表 6

图1 透视电子显微镜观察外膜囊泡OMVs结构左：E. coli DH5α 的OMVs，右：转化了pET-22b的E. coli DH5α 的OMVs

Fig. 1 Structure of OMVs observed by transmission electron microscopy Left ：OMVs of E. coli DH5α. Right ：OMVs of E. coli DH5α transformed with pET-22b

图2 NTA 检测OMVs的粒径分布 A：E. coli DH5α 的OMVs；B：转化了pET-22b的E. coli DH5α 的OMVs

Fig. 2 Particle size distribution of OMVs detected by NTA A: OMVs of E. coli DH5α ; B: OMVs of E. coli DH5α transformed with pET-22b

图3 WB检测外膜囊泡OMVs的外膜蛋白OmpC 1 ：E. coli DH5α 的OMVs；2：转化了pET-22b的E. coli DH5α 的OMVs

Fig. 3 Detection of outer membrane protein OmpC of OMVs by WB 1 : OMVs of E. coli DH5α; 2: OMVs of E. coli DH5α transformed with pET-22b

图4 OMVs中AmpR的PCR检测 1 -3：E. coli DH5α 的OMVs; 4-6：pET-22b载体-E. coli DH5α 的 OMVs; 7-9：无菌水；10-12：pET-22b载体；M：marker DL2000

Fig. 4 Detection of AmpR in OMVs by PCR 1 -3: OMVs of E. coli DH5α ; 4-6: OMVs of the pET-22b vector-E. coli DH5α; 7-9: sterile water;10-12: pET-22b vector; M: marker DL2000

图5 OMVs与无抗性E. coli DH5α 共孵育结果 A：空白组；B：10 μg OMVs（AmpR-）组：C：20 μg OMVs（AmpR-）组；D：10 μg OMVs-AmpR组；E：20 μg OMVs-AmpR组

Fig. 5 Co-incubation of OMVs with non-resistant E. coli DH5α A: Control group; B: 10 μg OMVs（AmpR-）group: C: 20 μg OMVs（AmpR-）group; D: 10 μg OMVs-AmpR group; E: 20 μg OMVs-AmpR group

图6 菌落PCR结果 1-3：空白组； 4-6: E. coli DH5α ；7-9：转化了pET-22b载体的E. coli DH5α；10-12：10 μg OMVs-AmpR组；13-15：20 μg OMVs-AmpR组；M：marker DL 2000

Fig. 6 Colony by PCR 1-3: Control group; 4-6: E. coli DH5α; 7-9: E. coli DH5α transformed with pET-22b vector; 10-12: 10 μg OMVs-AmpR group; 13-15: 20 μg OMVs-AmpR group; M: marker DL 2000

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