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A kind of Pseudomonas aeruginosa vaccine recombinant protein SBP and its preparation method and application

A Pseudomonas aeruginosa and recombinant protein technology, applied in chemical instruments and methods, recombinant DNA technology, antibacterial drugs, etc., can solve the problem of the absence of Pseudomonas aeruginosa vaccines, the increase in the isolation rate of drug-resistant PA, etc. problems, achieve the effect of maintaining spatial conformation and immunogenicity, good protection effect, and mild purification conditions

Active Publication Date: 2021-08-24
重庆艾力彼生物科技有限公司
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  • Abstract
  • Description
  • Claims
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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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  • A kind of Pseudomonas aeruginosa vaccine recombinant protein SBP and its preparation method and application
  • A kind of Pseudomonas aeruginosa vaccine recombinant protein SBP and its preparation method and application
  • A kind of Pseudomonas aeruginosa vaccine recombinant protein SBP and its preparation method and application

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preparation example Construction

[0036] like 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 th...

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 antigens, and discloses a recombinant protein SBP of Pseudomonas aeruginosa vaccine and its preparation method and application; the nucleotide sequence is SEQ ID NO: 1; the amino acid sequence is SEQ ID NO: 2; the recombinant The expression vector comprises the nucleotide sequence of SEQ ID NO:1. The present invention adopts pGEX-6p-1 carrier to construct recombinant expression plasmid, expresses recombinant protein SBP, pGEX is the carrier expressing fusion protein constructed by Smith and Johnson in 1987, and its main feature is that the carrier is connected with a molecular weight of 26kDa Glutathione‑S‑transferase (GST), the expressed fusion protein contains a GST tag, which is a marker for protein purification. Compared with other fusion vectors, the pGEX series vectors can keep the purified protein in its spatial conformation and immunogenicity to the greatest extent.

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 Patents(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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