Pseudomonas aeruginosa vaccine recombinant protein reSBP-ExoU, preparation method and application

A technology of Pseudomonas aeruginosa and recombinant protein, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as poor adjuvant adsorption and reExoU instability, simplify the production process, facilitate Separation and purification, the effect of reducing production costs

Active Publication Date: 2020-03-27
重庆艾力彼生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It was further found that the recombinant protein reSBP-ExoU not only solved the problems of poor adsorption between reSBP protein and human adjuvant and the instability of reExoU, but also had good immunogenicity and immune protection effect, which is of great significance for the next step in the development of Pseudomonas aeruginosa Immunomodulatory means such as bacterial vaccines are of great significance

Method used

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  • Pseudomonas aeruginosa vaccine recombinant protein reSBP-ExoU, preparation method and application
  • Pseudomonas aeruginosa vaccine recombinant protein reSBP-ExoU, preparation method and application
  • Pseudomonas aeruginosa vaccine recombinant protein reSBP-ExoU, preparation method and application

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Experimental program
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Embodiment approach

[0041] According to a preferred embodiment of the present invention, the recombinant protein has a 26kDa glutathione-S-transferase (GTS) tag attached to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, the The use of tags makes purification conditions mild, steps simple, and does not require the addition of denaturants, so that the purified protein can maintain its spatial conformation and immunogenicity to the greatest extent.

[0042] The above-mentioned recombinant protein can be obtained by artificial synthesis, or its coding gene can be synthesized first, and then obtained by biological expression.

[0043] In the second aspect, the present invention also provides a gene capable of encoding the above-mentioned recombinant protein.

[0044] It is well known in the art that among the 20 different amino acids that make up proteins, except that Met (ATG) or Trp (TGG) are encoded by single codons respectively, the other 18 amino acids are encoded by 2-6 codons ...

Embodiment 1

[0069] This example is used to illustrate the induction, purification and identification of the expression form of the recombinant fusion protein reSBP-ExoU in the prokaryotic expression system-Escherichia coli

[0070] (1) Synthesis and subcloning of the gene encoding reSBP-ExoU

[0071] The synthesis of the DNA sequence of reSBP-ExoU (SEQ ID NO: 2) and the connection of the DNA sequence of reSBP-ExoU and pGEX-6p-2 were synthesized by Shanghai Sangon Bioengineering Co., Ltd., thereby obtaining the recombinant plasmid pGEX-6p-reSBP- ExoU.

[0072] (2) Transformation and double enzyme digestion identification of recombinant plasmids

[0073] 1) Transformation of recombinant plasmids Escherichia coli XL1blue competent cells (Shanghai Chaoyan Biotechnology Co., Ltd.) were taken from a -80°C refrigerator, and 1 μl of recombinant plasmid pGEX-6p-reSBP-ExoU was added. Ice bath for 10 min, heat shock in a 42°C metal bath for 60 s, and quickly ice bath for 1 min. Add 600 μl of LB b...

Embodiment 2

[0090] This example is used to illustrate the preparation of reSBP-ExoU antigen

[0091] (1) Amplified culture to obtain protein

[0092] Take 400 μL of the spare pGEX-6P-2-reSBP-ExoU / XL-1blue bacterial solution stored in a refrigerator at 4°C and add it to 20 mL of LB medium containing Amp resistance for primary activation. After culturing at 200 rpm at 37°C for 5-6 hours, Take 8 mL of the primary activated bacterial liquid and add it to 400 mL of LB medium containing Amp resistance for secondary activation, culture at 37°C for 3-4 hours until the OD600 is 0.6-0.8, add 80 μL of IPTG (final concentration is 200 μM) in the After induction in a shaker at 16°C for 16 hours, centrifuge at 12,000 rpm for 15 minutes to collect the bacteria, add 20 mL of lysis buffer (same as Example 1) to resuspend the bacteria, and ultrasonically lyse the bacteria for 15 minutes, collect the supernatant and 800 μL for fusion The Glutathione Sepharose4B (GE Company) filler (beads) binding treatment...

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Abstract

The invention relates to the technical field of bacterial antigens, and discloses a pseudomonas aeruginosa vaccine recombinant protein reSBP-ExoU, a preparation method and application. An amino acid sequence of the vaccine recombinant protein is shown in SEQ ID NO.1. The recombinant protein reSBP-ExoU is prepared by fusing and connecting part of a SBP protein (Ala22-Phe260) and part of a non-toxicExoU mutant (Ser100-Asn360, S142A and Asp344Ala) through a flexible Linker-GSGGSG molecule; and the recombinant protein reSBP-ExoU not only solves the problem of poor adsorption of a reSBP protein toadjuvants applicable to human bodies and the problem of unstable reExoU, but also has good immunogenicity and immunoprotection effects, and thus has important significance on developing immunoregulation means such as pseudomonas aeruginosa vaccines next.

Description

technical field [0001] The present invention relates to the technical field of bacterial antigens, in particular to a Pseudomonas aeruginosa vaccine recombinant protein, a gene encoding the vaccine recombinant protein, an expression vector inserted with the gene, and a host containing the gene or vector Application of the cell, the vaccine recombinant protein or the gene or the expression vector or the host cell in the preparation of a drug for preventing and / or treating Pseudomonas aeruginosa infection, a kind of Pseudomonas aeruginosa vaccine , the use of the vaccine recombinant protein or the gene or the expression vector or the host cell in the preparation of anti-Pseudomonas aeruginosa antibodies, a method for producing the vaccine recombinant protein according to claim 1. Background technique [0002] Pseudomonas aeruginosa (PA) is the most common pathogen in nosocomial infections such as burns, war trauma, and mechanically ventilated patients, and can cause severe pne...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/21C12N15/31C12N15/70C12N1/21A61K39/104A61P31/04C12R1/19
CPCC07K14/21C12N15/70A61K39/104A61P31/04
Inventor 郭刚冯强张欣熊蜂张娇娇卢文根杨念
Owner 重庆艾力彼生物科技有限公司
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