Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Pseudomonas aeruginosa recombinant protein vac11 and its preparation method and application

A Pseudomonas aeruginosa and recombinant protein technology, applied in biochemical equipment and methods, recombinant DNA technology, chemical instruments and methods, etc., can solve the problem of decreased virulence of Pseudomonas aeruginosa bacteria and achieve good immune protection Effect, easy separation and purification, high expression effect

Active Publication Date: 2019-11-29
CHENGDU OLYMVAX BIOPHARM +1
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the deletion of the AmpDh3 gene will lead to a significant decrease in the virulence of Pseudomonas aeruginosa bacteria (Moya B, et al. AntimicrobAgents Chemother.2008)

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
  • Pseudomonas aeruginosa recombinant protein vac11 and its preparation method and application
  • Pseudomonas aeruginosa recombinant protein vac11 and its preparation method and application
  • Pseudomonas aeruginosa recombinant protein vac11 and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Cloning of Vac11 Gene and Construction of Recombinant Plasmid pGEX-6P-2-Vac11

[0046] 1. According to the full-length gene sequence of AmpDh3 protein of Pseudomonas aeruginosa PA01, the bioinformatics software was used for structural analysis to determine the target gene fragment of Vac11 to be amplified.

[0047] 2. According to the analysis results, the PCR method was used to amplify the AmpDh3 target gene fragment from the PA01 genome, and the amplification steps were as follows:

[0048] 1) Design the PCR primers as follows, respectively SEQ ID NO: 3-4 (the base sequence of the restriction site is underlined)

[0049]

[0050]

[0051] 2) Take out the preserved Pseudomonas aeruginosa strain PA01 from the freezer at -80°C and spread it on LB solid medium, culture it at 37°C overnight, then pick a single colony and inoculate it in LB liquid medium for 8 hours , referring to the bacterial genome extraction kit to extract the PA01 genome.

[0052] ...

Embodiment 2

[0080] Example 2: Induced expression, purification and identification of expression form of recombinant fusion protein Vac11 in prokaryotic expression system-Escherichia coli

[0081] 1. Vac11 induced expression

[0082] Take 100 μL of the overnight cultured pGEX-6P-2-Vac11 / XL-1blue bacterial solution and add it to 10 mL of Amp+ resistant LB medium, culture overnight at 180 rpm at 37°C, respectively take 400 μL of the overnight cultured bacterial solution and add 20 mL of Amp+ resistant LB medium In the culture medium (the rest of the bacterial solution is stored in a refrigerator at 4°C for later use), culture at 37°C for 2-3 hours, rotate at 200 rpm, and when the secondary activation reaches OD600 of 0.8-1.0, add 4 μL of IPTG to make the final concentration 200 μM, then place Induce expression on a shaking table at 30°C for 3h.

[0083] 2) Take out the bacterial solution after induced expression, centrifuge at 12000rpm for 5min, discard the supernatant, add 1mL lysisbuffer ...

Embodiment 3

[0090] Embodiment 3: Preparation of Vac11 antigen

[0091] 1. Amplify culture to obtain protein

[0092] Take 400 μL of the spare pGEX-6P-2-Vac11 / XL-1blue bacterial solution stored in a 4°C refrigerator and add it to 20 mL of LB medium containing Amp resistance for one activation. Add the primary activated bacterial solution to 400mL LB medium containing Amp resistance for secondary activation, culture at 37°C for 3-4h until the OD600 is 1.0, add 80μL IPTG (final concentration 200μM) and place in a shaker at 16°C After overnight induction, centrifuge at 12000rpm for 15min to collect the bacteria, add 20mL lysis buffer (same as in Example 2) to resuspend the bacteria, then ultrasonically lyse the bacteria for 3min (200V), collect the supernatant and 800μL for binding to the GST fusion protein Glutathione Sepharose 4B (GE Company) gel beads (beads) binding treatment; then SDS-PAGE gel electrophoresis.

[0093] 2. Use the enzyme digestion method to separate the target protein f...

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 provides a pseudomonas aeruginosa recombinant protein Vac11 as well as a preparation method and an application. Animal tests prove that the recombinant protein can effectively stimulate organisms to carry out higher humoral immune response and give play to good immunoprotection effects, thus being beneficial to prevention, diagnosis and treatment of pseudomonas aeruginosa. The recombinant protein prepared by adopting the method provided by the invention has high expression quantity and is convenient to separate and purify.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmacy, and specifically relates to a recombinant protein Vac11 (AmpDh3) of Pseudomonas aeruginosa, and further relates to a preparation method and application of the recombinant protein. Background technique [0002] Pseudomonas aeruginosa (PA), commonly known as Pseudomonas aeruginosa, is a non-fermenting bacteria of the genus Pseudomonas, widely distributed in nature, normal human skin, intestinal tract, respiratory tract, and in hospital wards and medical equipment It is also ubiquitous and is one of the most common opportunistic pathogens in clinical practice. At present, the bacterium has become one of the pathogenic bacteria with the highest isolation rate in ICU wards, burns, war trauma infections, and mechanical ventilation-associated pneumonia (VAP) worldwide. PA infection can occur in any part and tissue of the human body, such as burns or trauma, middle ear, cornea, urethra, and res...

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
Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21G01N33/68G01N33/569A61K39/104A61P31/04C12R1/385
CPCA61K39/104A61K2039/54A61K2039/55505C07K2319/00C12N9/6489C12N15/62C12N15/70C12N2800/101C12Y304/24G01N33/56911G01N33/68G01N2333/21
Inventor 章金勇顾江邹全明杨峰杨茜赵莉群彭六生吴翼王逸麟
Owner CHENGDU OLYMVAX BIOPHARM
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com