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

A technology of Pseudomonas aeruginosa and recombinant protein, applied in chemical instruments and methods, recombinant DNA technology, antibacterial drugs, etc., can solve the problems of increased isolation rate of drug-resistant PA and difficulty of ExoU, and achieve good resistance to PA infection , good protection effect, low cost effect

Active Publication Date: 2021-08-24
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

[0004] 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
However, due to the particularity and complexity of the molecular structure of ExoU, it is very difficult to directly prepare natural, non-toxic ExoU in genetically engineered bacteria under the current technical conditions.
Therefore, it is only possible to realize the expression of ExoU in genetic engineering by modifying the natural ExoU protein at the gene level by truncation, mutation, etc., but there is no non-toxic ExoU recombinantly expressed in E. coli. Mutants, and use as candidate vaccine antigens

Method used

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  • A kind of recombinant protein rexou of Pseudomonas aeruginosa vaccine and its preparation method
  • A kind of recombinant protein rexou of Pseudomonas aeruginosa vaccine and its preparation method
  • A kind of recombinant protein rexou of Pseudomonas aeruginosa vaccine and its preparation method

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preparation example Construction

[0030] like figure 1 As shown, the preparation method of the Pseudomonas aeruginosa vaccine recombinant protein rExoU provided in the embodiments of the present invention includes the following steps:

[0031] S101: Take 400 μL of the spare pGEX-6p-1-rExoU / 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, and culture at 200 rpm for 5-6 hours at 37°C. Take 10mL of the primary activated bacterial liquid and add it to 1000mL LB medium containing Amp resistance for secondary activation;

[0032] S102: Cultivate at 37°C for 3-4 hours until OD600 is 1.0, add 200 μL IPTG (final concentration: 200 μM) and place in a shaker at 16°C overnight for induction, then centrifuge at 12,000 rpm for 15 minutes to collect the bacteria, then add 50 mL of lysis buffer: after resuspending the bacteria , the bacterial liquid was ultrasonically lysed for 3min (200V), and the supernatant was collected to be combin...

Embodiment 1

[0040] Example 1: Synthesis and subcloning of genes

[0041] 1. Synthesis of rExoU-encoding DNA (SEQ ID NO: 1) and sequence connection with pGEX-6p-1 was synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0042] 2. Transformation of recombinant plasmids Take 3 tubes of Escherichia coli XL1blue competent cells (Shanghai Chaoyan Biotechnology Co., Ltd.) from the -80°C refrigerator, and add pGEX-6P-1 plasmid (GE Healthcare LifeSciences) to the first tube as a positive control; Add 1 μl of synthetic pGEX-6P-rExoU plasmid to the second tube; no exogenous DNA is added to the third tube as a negative control. Ice bath for 50min, heat shock in 42℃ metal bath for 90s, rapid ice bath for 2min. Add 600 μl LB blank medium, mix well, and place in a shaker at 37°C at 220rpm for 1h.

[0043] Each tube was centrifuged at 5000 rpm for 3 min at room temperature, discarded 300 μl of supernatant, and then resuspended the bacteria, took 200 μl and spread it on an Amp-resistant LB plate. ...

Embodiment 2

[0049] Example 2: Induced expression, purification and identification of expression form of recombinant fusion protein rExoU in prokaryotic expression system-Escherichia coli

[0050] 1. rExoU induced expression

[0051] Take 100 μL of the overnight cultured pGEX-6p-1-rExoU / 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 culture medium (the rest of the bacterial solution is stored in a refrigerator at 4°C for later use), cultured at 37°C for 2-3 hours at a rotation speed of 200 rpm, and when the second activation was performed until the OD600 was 0.6-0.8, 4 μL (1mol / L) of IPTG was added to make the final concentration to 200 μM, and then placed on a shaker to induce expression at 30°C for 3 hours.

[0052] 2) Take out the bacterial solution after induced expression, centrifuge at 12000rpm for 5min, discard ...

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Abstract

The invention belongs to the technical field of bacterial antigens, and discloses a recombinant protein rExoU of a Pseudomonas aeruginosa vaccine and a preparation method thereof. The amino acid sequence is SEQ ID NO:2; the DNA coding sequence is SEQ ID NO:1. Amplify the culture to obtain the protein; use the enzyme digestion method to separate the target protein from the GST tag to obtain the rExoU target protein. The immune response induced by rExoU has a good protective effect against PA infection. Recombinant rExoU protein can be expressed in prokaryotic expression system - Escherichia coli, with low cost and high yield; when choosing pGEX vector series, rExoU recombinant protein is expressed in the form of fusion protein; the expression vector is connected with a glutathione-glutathione with a molecular weight of 26kDa S-transferase, the expressed fusion protein contains a GST tag, so that the purified rExoU protein can maintain 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 rExoU of a Pseudomonas aeruginosa vaccine and a preparation method thereof. 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, cornea, u...

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 郭刚罗莉周璐杨念郑艮梅
Owner 重庆艾力彼生物科技有限公司
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