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Escherichia coli Outer Membrane Vesicle Immunization Protocol and Induction of Bacterial Sepsis
利用大肠杆菌外膜囊泡诱发细菌脓毒症的模型   

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Abstract

Outer membrane vesicles (OMVs) are spherical bilayered phospholipids of 20-200 nm in size produced from all Gram-negative bacteria and Gram-positive bacteria investigated to date. OMVs, which resemble the outer membrane and periplasm in composition, are proinflammatory and immunogenic facsimiles, and therefore could activate both innate and adaptive immunity. Here, we describe the OMVs immunization protocol and bacteria challenge protocol to induce bacterial sepsis in mice.

Keywords: Outer membrane vesicles(外膜囊泡), Sepsis(脓毒症), Animal model(动物模型), Bacterial extracellular vesicles(细菌胞外囊泡), Immunization(免疫)

Materials and Reagents

  1. Five-week-old male C57BL/6 or BALB/c mice or a variety of transgenic/knockout mice, body weight 18-20 g
  2. Phosphate buffered saline (PBS) (Gibco®, catalog number: 70013-032 )
  3. Escherichia coli (E. coli) derived OMVs
  4. E. coli (Isolated from the peritoneal lavage fluid of cecal ligation and puncture-operated mice)
  5. Luria-Bertani broth (LB) medium (Merck KGaA, catalog number: 1.10285.0500 )

Equipment

  1. 1.5 ml microtube
  2. Ultra-fine-II insulin syringe 1 ml 31 G (0.25 mm x 8 mm) (BD, catalog number: 328820 )

Procedure

  1. Vaccine preparation
    1. Prepare a suspension containing 10 μg/ml of E.coli OMVs in PBS.
       Notes:
      1. The total amount of the sample depends on the number of mice in the immunized group. For each mouse, 100 μl of sample will be given. Add extra 100 μl to compensate for any loss of sample during each injection. i.e. If N=5 per group, make a total of 600 μl for each group.
      2. Refer to the protocol " Preparation of Out Membrane Vesicle from Escherichia coli " (Kim et al., 2013b) for preparing OMVs.
    2. Make three aliquots of the above OMVs sample in each 1.5 ml microtube and store at -80 °C until use.

  2. Immunization
    1. At day 0, label the cage (or mouse) to distinguish between immunized and sham.
    2. Bring one of the OMVs sample aliquot to room temperature and mix by vortexing before use.
    3. Hold the mouse in your hand by the dorsal skin so that the head of the mice is pointing the top and its rear legs are down. Maintain the tail with the fingers as in Figure 1 below.


      Figure 1. Intraperitoneal injection

  3. Bacteria challenge for induction of sepsis
    1. At day 21, prepare E. coli (5 x 109 CFU/ml) in PBS after culturing E. coli in LB broth for 200 rpm at 37 °C until OD600=1.0.
      Note: The total amount of the sample depends on the number of mice that will be challenged. For each mouse, 200 μl of the sample will be given. Add extra 100 μl to compensate for any loss of sample during each injection. i.e. For a total of 10 mice, make 2,100 μl of the sample.
    2. Hold the mouse in your hand by the dorsal skin so that the head of the mice is pointing the top and its rear legs are down. Maintain the tail with the fingers.
    3. Use sterile 1 ml syringe to intraperitoneally inject 200 μl of the E. coli suspension per mouse.
    4. Monitor the survival of mice every 12 h and record.
      Note: The symptoms for sepsis will appear after about 6-8 h of challenge. Sacrifice the mice after 5 days of monitoring. Please refer to the reference article for survival results.

Acknowledgments

This protocol was adapted from previously published work (Kim et al., 2013a). The work was supported by a grant from the Korean Ministry of Education, Science and Technology, FPR08B1-240 of the 21C Frontier Functional Proteomics Program and Mid-career Researcher Program of National Research Foundation of Korea (NRF) grant funded by the Korea government MEST (No. 20110000215 and No. 20120005634).

References

  1. Kim, O. Y., Hong, B. S., Park, K. S., Yoon, Y. J., Choi, S. J., Lee, W. H., Roh, T. Y., Lotvall, J., Kim, Y. K. and Gho, Y. S. (2013a). Immunization with Escherichia coli outer membrane vesicles protects bacteria-induced lethality via Th1 and Th17 cell responses. J Immunol 190(8): 4092-4102.
  2. Kim, O. Y., Hong, B. S., Park, K. S., Yoon, Y. J., Choi, S. J., Lee, W. H., Roh, T. Y., Lotvall, J., Kim, Y. K. and Gho, Y. S. (2013b). Preparation of outer membrane vesicle from Escherichia coli. Bio-protocol 3(23): e955.

简介

外膜囊泡(OMV)是从所有革兰氏阴性细菌和革兰氏阳性细菌产生的20-200nm的球形双层磷脂,迄今为止研究。 在组成上类似于外膜和周质的OMV是促炎和免疫原性传真,因此可以激活先天免疫和适应性免疫。 在这里,我们描述OMVs免疫协议和细菌攻击协议,以诱导小鼠的细菌性败血症。

关键字:外膜囊泡, 脓毒症, 动物模型, 细菌胞外囊泡, 免疫

材料和试剂

  1. 5周龄雄性C57BL/6或BALB/c小鼠或各种转基因/敲除小鼠,体重18-20g
  2. 磷酸盐缓冲盐水(PBS)(Gibco ,目录号:70013-032)
  3. 大肠杆菌(大肠杆菌)衍生的OMV
  4. E。 大肠杆菌(从盲肠结扎和穿刺操作的小鼠的腹膜灌洗液中分离)
  5. Luria-Bertani肉汤(LB)培养基(Merck KGaA,目录号:1.10285.0500)

设备

  1. 1.5 ml微量管
  2. 超细II胰岛素注射器1ml 31G(0.25mm×8mm)(BD,目录号:328820)

程序

  1. 疫苗制备
    1. 在PBS中制备含有10μg/ml大肠杆菌 OMV的悬浮液 注意:
      1.   样品的总量取决于小鼠的数量 免疫组。 对于每只小鼠,将给予100μl样品。 加 额外100μl,以补偿每次注射期间样品的任何损失。   即如果每组N = 5,每组总共600μl。
      2. 请参阅协议"从大肠杆菌制备膜囊泡"(Kim等人 。,2013b)以准备OMV。
    2. 在每个1.5 ml微量管中制备三份上述OMV样品,并储存于-80°C直至使用。

  2. 免疫
    1. 在第0天,标记笼(或小鼠)以区分免疫和假手术。
    2. 将其中一个OMV样品等分试样加入室温,并在使用前通过涡旋混合。
    3. 保持   鼠标在你手中的背部皮肤使得头部的小鼠 指向顶部,它的后腿向下。 保持尾巴 手指如下图1所示

      图1.腹膜内注射

  3. 诱导败血症的细菌攻击
    1. 在第21天,准备E。 在LB肉汤中在37℃下培养大肠杆菌200rpm后在PBS中培养大肠杆菌(5×10 9 CFU/ml),直到OD < 600 = 1.0。
      注意:   样品的总量取决于小鼠的数量 受到挑战。 对于每只小鼠,将给予200μl样品。 加 额外100μl,以补偿每次注射期间样品的任何损失。   即对于总共10只小鼠,制备2,100μl样品
    2. 保持 鼠标在你手中的背部皮肤使得头部的小鼠 指向顶部,它的后腿向下。 保持尾巴 手指。
    3. 使用无菌1毫升注射器腹腔注射200微升的E。 大鼠悬浮液。
    4. 每12小时监测小鼠的存活并记录 注意:   脓毒症的症状将在约6-8小时的攻击后出现。 在监测5天后牺牲小鼠。 请参考 参考文章的生存结果。

致谢

此协议改编自以前发表的作品(Kim等人,2013a)。这项工作得到了韩国教育,科学技术部的资助,21C前沿功能蛋白质组学项目的FPR08B1-240和韩国国家研究基金会(NRF)的中期职业生涯研究员资助由韩国政府MEST资助(No.20110000215和No.20120005634)。

参考文献

  1. Kim,O.Y.,Hong,B.S.,Park,K.S.,Yoon,Y.J.,Choi,S.J.,Lee,  H.,Roh,T.Y.,Lotvall,J.,Kim,Y.K.and Gho,Y.S。(2013a)。 用大肠杆菌外膜囊免疫可通过Th1保护细菌诱导的致死性和Th17细胞应答。 J Immunol 190(8):4092-4102。
  2. Kim,O.Y.,Hong,B.S.,Park,K.S.,Yoon,Y.J.,Choi,S.J.,Lee,  H.,Roh,TY,Lotvall,J.,Kim,YK and Gho,YS(2013b)。制备外膜囊泡大肠杆菌 生物协议 3(23):e955。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kim, O. Y., Hong, B. S., Park, K., Yoon, Y. J., Choi, S. J., Lee, W. H., Roh, T., Kim, Y. and Gho, Y. S. (2013). Escherichia coli Outer Membrane Vesicle Immunization Protocol and Induction of Bacterial Sepsis. Bio-protocol 3(23): e994. DOI: 10.21769/BioProtoc.994.
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