Extraction method for outer membrane vesicles of salmonella typhimurium
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A Salmonella and outer membrane vesicle technology, applied in the field of extraction of Salmonella typhimurium outer membrane vesicles
Active Publication Date: 2019-04-02
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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[0004] In order to solve the problems existing in the prior art, the present invention provides a method for extracting outer membrane vesicles of Salmonella typhimurium, which solves the problem of ultracentrifugation, and the high-speed centrifugation scheme applied in the present invention successfully uses a centrifugal force of 40,000×g The OMVs of Salmonella typhimurium were extracted
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Embodiment 1
[0040] 1. Extraction of OMVs from Salmonella typhimurium
[0041] 1. Centrifuge 1L of Salmonella CVCC541 cultured to the early stage of the stationary phase at 10,000×g, 4°C, for 15 minutes.
[0042] 2. Recover the supernatant, and be careful not to pour out the precipitated bacterial solution. You can keep a certain amount of liquid and perform centrifugation at 13,000×g, 4°C, for 15 minutes.
[0043] 3. Recover the supernatant, and filter twice with a 0.45 μm PVDF membrane filter.
[0044] 4. The filtered supernatant was subjected to 40,000×g, 4°C, 2h. Due to the limited size of the centrifuge tube, 1 L of filtrate can be centrifuged several times.
[0045] 5. Discard the supernatant, resuspend and dilute with 0.01M PBS. And it was filtered with a 0.45 μm PVDF filter, and 1 ml of the filtrate was taken to plate to identify whether the bacteria were completely removed.
[0046] The formula of 0.01M PBS is: 8g NaCl, 0.2g KCl, 1.42g Na 2 HPO 4 and 0.27g KH 2 PO 4 , diss...
Embodiment 2
[0060] In order to eliminate a large amount of flagellar components in the OMVs extract obtained in Example 1 and improve the purity of the OMVs, different methods were used to process it.
[0061] 1. The obtained OMVs extract was further purified by density gradient centrifugation to remove flagella
[0062] The OMVs extract extracted in Example 1 was added to the Optiprep reagent of 45%, 40%, 35%, 30%, 25%, and 20% content from bottom to top, and ultracentrifugation was carried out at 10000g, 4°C, and 16h, Take out the whole volume of the centrifuge from top to bottom, and perform SDS-PAGE electrophoresis for protein electrophoresis to observe the purification results. see results Figure 4 .
[0063] Figure 4 Analysis of the presence of OMVs for density gradient centrifugation. From left to right are M and lanes 1-12. Among them, M: Marker; 1, 2: 20% Optiprep; 3, 4: 25% Optiprep; 5, 6: 30% Optiprep; 7, 8: 35% Optiprep; 9, 10: 40% Optiprep; 11, 12: 45% Optiprep.
[0...
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Abstract
The present invention provides an extraction method for outer membrane vesicles (OMVs) of salmonella typhimurium. The extraction method comprises the following steps: (1) salmonella typhimurium liquidis subjected to centrifuging at 10,000 x g and 4 DEG C for 15 min; (2) a supernatant is recovered, and a salmonella typhimurium liquid precipitate is not poured out and subjected to centrifuging at 13,000 x g and 4 DEG C for 15 min; (3) the supernatant is recovered and filtering is conducted twice with a PVDF membrane filter; and (4) the filtered supernatant is subjected to centrifuging at 40,000x g and 4 DEG C for 2 h; and the supernatant is discarded. The applied high-speed centrifugation scheme successfully extracts the OMVs of the salmonella typhimurium with a centrifugal force of 40,000x g. The extraction method also solves a problem that flagella are abundantly present in the OMVs extract. A gene targeting editing technique is applied to knock out flagella genes of the salmonellatyphimurium to obtain the high-purity OMVs.
Description
technical field [0001] The invention relates to a method for extracting outer membrane vesicles of Salmonella typhimurium. Background technique [0002] Outer membrane vesicles (Outer membrane vesicles, OMVs) have a nanoscale membrane structure of lipid bilayers, with a size between 20-250nm, and are naturally produced by Gram-negative bacteria. Both pathogenic and non-pathogenic bacteria can produce OMVs, which contain a large number of bacterial components, including lipopolysaccharide (LPS), periplasmic and membrane-associated proteins, enzymes, toxins, DNA, RNA and peptidoglycan. The way of formation is mainly due to protein misfolding or the destruction of the outer membrane structure, the outer membrane forms bulges and is exported in the form of buds. Because its production is related to bacterial drug resistance, pathogenicity, biofilm formation, etc., and can be used as a new biological carrier, its research has become a hot spot in recent years. [0003] The prim...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/21C12R1/42
CPCC12N1/20C07K14/255
Inventor 敬文宪刘永生李学瑞周建华马丽娜
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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