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Pseudomonas aeruginosa vaccine recombinant protein SBP, and preparation method and applications thereof

A Pseudomonas aeruginosa, recombinant protein technology, applied in recombinant DNA technology, chemical instruments and methods, antibacterial drugs, etc. problem, to achieve good anti-PA infection, good protection effect, easy separation and purification effect

Active Publication Date: 2019-02-01
重庆艾力彼生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In summary, the problems in the prior art are: due to the abuse of antibiotics and other reasons, the problem of PA drug resistance has gradually become prominent, and pan-drug-resistant Pseudomonas aeruginosa (PDR-PA) and multidrug-resistant Pseudomonas aeruginosa have appeared. bacteria (MDR-PA), and the isolation rate of drug-resistant PA is increasing year by year
From the perspective of immunology, it is the most ideal choice to develop a safe and effective vaccine, but there is no Pseudomonas aeruginosa vaccine on the market.

Method used

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  • Pseudomonas aeruginosa vaccine recombinant protein SBP, and preparation method and applications thereof
  • Pseudomonas aeruginosa vaccine recombinant protein SBP, and preparation method and applications thereof
  • Pseudomonas aeruginosa vaccine recombinant protein SBP, and preparation method and applications thereof

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preparation example Construction

[0036] Such as figure 1 As shown, the preparation method of the Pseudomonas aeruginosa vaccine recombinant protein SBP provided by the embodiments of the present invention comprises the following steps:

[0037] S101: Take 400 μL of the spare pGEX-6p-1-SBP / XL-1blue bacterial solution stored in a refrigerator at 4°C and add it to 20 mL of LB medium containing Amp resistance for one activation. After culturing at 200 rpm and 37°C for 5-6 hours, Take 10mL of the primary activated bacterial liquid and add it to 1000mL LB medium containing Amp resistance for secondary activation, and culture at 37°C for 3-4h until OD600 is 1.0;

[0038] S102: After adding 200 μL IPTG (final concentration of 200 μM) and placing it in a shaker at 16° C. for overnight induction, centrifuge at 12,000 rpm for 15 minutes to collect the bacterial cells, add 50 mL of lysis buffer (same as Example 2) to resuspend the bacterial cells, and then ultrasonicate the bacterial liquid Lyse for 3min (200V), collect...

Embodiment 1

[0051] Embodiment 1: The cloning of SBP gene and the construction of recombinant plasmid pGEX-6P-1-SBP

[0052] 1. Firstly, according to the full-length gene sequence of the SBP protein of Pseudomonas aeruginosa PA01, bioinformatics software was used for structural analysis to determine the SBP target gene fragment to be amplified.

[0053] 2. According to the analysis results, the PCR method was used to amplify the SBP target gene fragment from the PA01 genome, and the amplification steps were as follows:

[0054] 1) Design the PCR primers as follows, respectively SEQ ID NO: 3-4 (the base sequence of the restriction site is underlined)

[0055]

[0056] 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.

[0057] 3) Using ...

Embodiment 2

[0084] Example 2: Induced expression, purification and identification of expression form of recombinant fusion protein SBP in prokaryotic expression system-Escherichia coli

[0085] 1. SBP induced expression

[0086] 1) Take 100 μL of the overnight cultured pGEX-6p-1-SBP / XL-1blue bacterial solution and add it to 10 mL of Amp+ resistant LB medium, culture overnight at 180 rpm at 37°C, take 400 μL of the overnight cultured bacterial solution and add 20 mL of Amp+ resistant LB medium (the rest of the bacterial solution was stored in a 4°C refrigerator for later use), cultured at 37°C for 2-3 hours at a rotation speed of 200 rpm, and when the secondary activation reached OD600 of 0.6-0.8, 4 μL (1mol / L) of IPTG was added to make The final concentration was 200 μM, and then placed on a shaker to induce expression at 30° C. for 3 hours.

[0087]2) Take out the bacterial solution after induced expression, centrifuge at 12000rpm for 5min, discard the supernatant, add 1mL of lysisbuffe...

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Abstract

The invention belongs to the technical field of bacterial antigen, and discloses a pseudomonas aeruginosa vaccine recombinant protein SBP, and a preparation method and applications thereof. The nucleotide sequence is SEQ ID NO:1; the amino acid sequence is SEQ ID NO:2; a recombinant expression carrier contains a nucleotide sequence SEQ ID NO:1. According to the preparation method, pGEX-6p-1carrieris adopted in construction of a recombinant expression plasmid for expression of recombinant protein SBP; pGEX is an expression fusion protein carrier constructed by Smith and Johnson in 1987, and the main characteristic is that the carrier is grafted with glutathione-S-transferase (GST) with a molecular weight of 26kDa; the expressed fusion protein contains a GST label; and the label is a protein purification label. Compared with other fusion carriers, the pGEX carrier is capable of maintaining the space conformation and the immunogenicity of the purified protein as far as possible.

Description

technical field [0001] The invention belongs to the technical field of bacterial antigens, and in particular relates to a recombinant protein SBP of Pseudomonas aeruginosa vaccine and its preparation method and application. Background technique [0002] At present, the commonly used prior art in the industry is as follows: Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA) is commonly called as Pseudomonas aeruginosa, is the Pseudomonas in the non-fermenting bacteria, is widely distributed in nature, normal human skin, intestinal tract It is also commonly found in hospital wards and medical equipment, and is one of the most common clinical opportunistic pathogens. 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, cor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/21C07K19/00C12N15/70A61K39/104A61P31/04
CPCA61K39/104A61P31/04C07K14/21C07K2319/23C12N15/70
Inventor 郭刚郑艮梅杨念周璐罗莉
Owner 重庆艾力彼生物科技有限公司
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