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A kind of Pseudomonas aeruginosa recombinant protein vac33 and its preparation method and application

A Pseudomonas aeruginosa and recombinant protein technology, applied in biochemical equipment and methods, chemical instruments and methods, recombinant DNA technology, etc., to achieve good immune protection effect, maintain spatial conformation and immunogenicity, and simple steps

Active Publication Date: 2019-11-08
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Similarly, PcrV is also an important candidate vaccine molecule, but due to its ability to form homologous multimers and other reasons, there have been no reports of soluble recombinant expression of this protein monomer.

Method used

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  • A kind of Pseudomonas aeruginosa recombinant protein vac33 and its preparation method and application
  • A kind of Pseudomonas aeruginosa recombinant protein vac33 and its preparation method and application
  • A kind of Pseudomonas aeruginosa recombinant protein vac33 and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction and Identification of Recombinant Plasmids

[0034] 1. The synthesis of the DNA sequence of Vac33 and the connection of the sequence and pGEX-6p-2 were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0035] 2. Transformation of recombinant plasmids

[0036] Take 3 tubes of Escherichia coli XL1blue competent cells (Shanghai Chaoyan Biotechnology Co., Ltd.) from the -80°C refrigerator, add pGEX-6P-2 plasmid (GE Healthcare Life Sciences) to the first tube as a positive control; add 1ul to the second tube Vac33 synthesized plasmid; the third tube without exogenous DNA was used 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 shake at 220rp for 1 hour in a shaker at 37°C.

[0037] 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 i...

Embodiment 2

[0043] Example 2 Induced expression, purification and identification of expression form of recombinant fusion protein PcrV-OprI in prokaryotic expression system-Escherichia coli

[0044] 1. Vac33 induced expression

[0045] Take 100 μL of the overnight cultured pGEX-6P-2-Vac33 / 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 it to 20 mL of Amp+ resistant LB medium medium (the rest of the bacterial solution was stored in a 4°C refrigerator for later use), cultured at 37°C for 2 to 3 hours at a rotational speed of 200rpm, and when the second activation was performed until the OD600 was 0.8-1.0, 4 μL of IPTG was added to make the final concentration 200 μM, and then placed in Induce expression on a shaking table and induce expression at 30°C for 3h.

[0046] 2) Take out the bacterial solution after induced expression, centrifuge at 12...

Embodiment 3

[0053] Example 3 Preparation of Vac33 Antigen

[0054] 1. Amplify culture to obtain protein

[0055] Take 400 μL of the spare pGEX-6P-2-Vac33 / 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 12,000rpm for 15min to collect the bacteria, add 20mL lysis buffer (same as 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.

[0056] 2. Use the enzyme digestion method to separate the target protein from th...

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Abstract

The invention provides recombinant protein. The recombinant protein is formed by fusion of a PA III type secretory system component protein (PcrV) fragment and a PA outer membrane lipoprotein (OprI) fragment through a Linker, and has a general formula: PcrV fragment-(Linker)n-Oprl fragment, wherein the sequence of the Linker is selected from one of GGGGS, GGSGG and YAPVDV, and preferentially GGGGS; n is 1, 2, 3 or 4, and preferentially 1. The invention also provides a preparation method and an application of the recombinant protein. Subunit vaccine prepared from the recombinant protein can stimulate the organism to generate high-titer IgG antibodies; the recombinant protein can be used for preparing diagnostic reagents, and preventive or therapeutic drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmacy, and specifically relates to a recombinant protein Vac33 (PcrV-OprI) 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/70A61K39/104A61P31/04G01N33/569
CPCA61K39/104A61K2039/54A61K2039/545C07K14/21C07K2319/00C12N15/70C12N2800/101G01N33/56911G01N2333/21
Inventor 顾江章金勇邹全明赵莉群杨茜杨峰彭六生吴翼王逸麟
Owner CHENGDU OLYMVAX BIOPHARM
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