Extraction method and application suitable for obtaining large amounts of high-purity salmonella outer membrane proteins

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 difficult separation of plasma membrane and outer membrane, cumbersome extraction process, and inapplicability Extraction of outer membrane protein and other issues to achieve the effect of improving efficiency and purity, improving purification efficiency, and convenient operation

Active Publication Date: 2019-02-22
SOUTH CHINA AGRI UNIV
View PDF7 Cites 2 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Extraction method and application suitable for obtaining large amounts of high-purity salmonella outer membrane proteins
  • Extraction method and application suitable for obtaining large amounts of high-purity salmonella outer membrane proteins
  • Extraction method and application suitable for obtaining large amounts of high-purity salmonella outer membrane proteins

Examples

Experimental program
Comparison scheme
Effect test

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses an extraction method and application suitable for obtaining large amounts of high-purity salmonella outer membrane proteins. The method includes the following steps: (1) cloning genes of the salmonella outer membrane proteins into an expression vector, and then introducing the obtained recombinant expression vector into host salmonella to obtain recombinant strains; (2) conducting enlargement cultivation on the recombinant strains, collecting the strains by centrifugation and conducting cryopreservation to obtain frozen strains; (3) adding protein lysate to the frozen strains, adding a phenylmethylsulfonyl fluoride solution, conducting constant temperature ultrasonication and collecting a protein precipitate; (4) adding an outer membrane protein extract to the protein precipitate, mixing evenly, incubating at 4 DEG C, then centrifuging and collecting a liquid supernatant; (5) separating and purifying the supernatant to obtain the salmonella outer membrane proteins. The method is simple in steps, convenient to operate and suitable for large-scale extraction of the high-purity high-activity outer membrane proteins and can simultaneously satisfy subsequent experimental requirements for protein activity, structure, vaccine development and the like.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap