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An extraction method suitable for obtaining a large amount of high-purity Salmonella outer membrane protein and its application

A technology of outer membrane protein and Salmonella, which is applied to the preparation method of peptides, chemical instruments and methods, medical preparations containing active ingredients, etc., can solve the problem of distinguishing the plasma membrane from the outer membrane, which is not suitable for extracting outer membrane proteins , cumbersome extraction process and other issues, to achieve the effect of improving efficiency and purity, convenient operation, and improving purification efficiency

Active Publication Date: 2021-02-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the methods commonly used to extract outer membrane proteins include sodium carbonate method, isopycnic gradient centrifugation method, Sarkosyl method, and Triton-X114 method. These methods have relatively high requirements for equipment, and the extraction process is relatively cumbersome. It is difficult to strictly distinguish the plasma membrane and the outer membrane, which belong to the same membrane structure
Therefore, existing methods are not suitable for the extraction of outer membrane proteins of Salmonella

Method used

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  • An extraction method suitable for obtaining a large amount of high-purity Salmonella outer membrane protein and its application
  • An extraction method suitable for obtaining a large amount of high-purity Salmonella outer membrane protein and its application
  • An extraction method suitable for obtaining a large amount of high-purity Salmonella outer membrane protein and its application

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Embodiment 1

[0054] The extraction of embodiment 1 Salmonella outer membrane protein

[0055] An extraction method suitable for obtaining a large amount of high-purity Salmonella outer membrane protein, comprising the following steps:

[0056] (1) Construction and introduction of expression vector: Oligo 7.0 was used to design primers for the construction of acrB gene (Primer F: 5'-TTTAAGAAGGAGATACATATGCCTAATTTCTTTATCGATCGC-3', Primer R: 5'-TGGTGGTGGTGCTCGAGGCGATGTTCTGTCGAATGACTA-3'), and synthesized by BGI . The genomic template of Salmonella typhimurium SL1344 (purchased from Beijing Kezhan Biotechnology Co., Ltd.) was obtained by boiling water, and used as a PCR template, using the high-fidelity enzyme Platinum TM Pfx DNA polymerase (Invitrogen) utilizes the above primers to amplify the acrB gene, constructs the gene into the pBad33 expression vector with a histidine tag (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) after enzyme digestion, and The constructed vector was int...

Embodiment 2

[0066] Example 2: Detection and verification of Salmonella outer membrane protein

[0067] The outer membrane protein obtained in Example 1 is detected, and the specific method is as follows:

[0068] (1) Detect protein with SDS-PAGE electrophoresis method, install the purchased 12% precast gel into the electrophoresis tank, add electrophoresis buffer to the short plate of the gel plate above 0.5cm, electrophoresis buffer (Tris-glycine buffer pH8.3 ) preparation: Weigh 6.0g Tris, 28.8g glycine, 1.0g SDS, dissolve in water and dilute to 1L.

[0069] (2) Mix the outer membrane protein obtained in Example 1 with 6×loding buffer at a volume ratio of 1:5, add it to the bottom of the concave sample tank of the gel, add the protein maker, and start electrophoresis. The electrophoresis voltage is 160V, and the electrophoresis time is for 40min.

[0070] (3) Take out the gel and place it in a petri dish, pour the staining solution into the petri dish, stain for about 1 hour, rinse wi...

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Abstract

The invention discloses an extraction method and application suitable for obtaining a large amount of high-purity Salmonella outer membrane protein. The method comprises the following steps: (1) cloning the gene of Salmonella outer membrane protein into an expression vector, then introducing the obtained recombinant expression vector into host Salmonella to obtain a recombinant strain; (2) expanding and culturing the recombinant strain, and then Collect the cells by centrifugation and cryopreserve to obtain the frozen cells; (3) add protein lysate and phenylmethylsulfonyl fluoride solution to the frozen cells, and ultrasonically break at constant temperature to collect protein precipitates; (4) add protein precipitates Add the outer membrane protein extract to the mixture, mix well and incubate at 4°C, then centrifuge to collect the supernatant; (5) separate and purify the supernatant to obtain the Salmonella outer membrane protein. The method of the invention has simple steps and convenient operation, is suitable for large-scale extraction of high-purity and high-activity outer membrane proteins, and can simultaneously meet the requirements of follow-up experiments such as protein activity, structure, and vaccine research and development.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an extraction method and application suitable for obtaining a large amount of high-purity Salmonella outer membrane protein. Background technique [0002] Salmonella is a zoonotic pathogen of great significance in public health. It is recognized by various countries, has the most reports in the world, and is the most common primary pathogen causing foodborne diseases in the world. It can cause diseases such as gastroenteritis, bacteremia and typhoid fever . Most members of the Salmonella genus are highly pathogenic. Salmonella belongs to the Enterobacteriaceae Salmonella genus. It is a Gram-negative bacterium with blunt ends and medium size. It has no spores and generally no capsule. Except for Salmonella pullorum and Salmonella gallinarum typhi, the rest have flagella all over the body. , motile, most ciliated, aerobic or facultative anaerobes. Salmonella most often a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C07K14/255C07K1/14A61K39/112A61P31/04
CPCA61K39/0275A61P31/04C07K14/255C12N15/74Y02A50/30
Inventor 曾振灵蒋红霞杨玲林晓玲史海洋
Owner SOUTH CHINA AGRI UNIV
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