PA (pseudomonas aeruginosa) recombinant protein POP, as well as preparation method and application thereof

A Pseudomonas aeruginosa, recombinant protein technology, applied in biochemical equipment and methods, chemical equipment and methods, recombinant DNA technology, etc., to achieve good immune protection effect, maintain spatial conformation and immunogenicity, and mild purification conditions Effect

Active Publication Date: 2016-07-06
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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  • PA (pseudomonas aeruginosa) recombinant protein POP, as well as preparation method and application thereof
  • PA (pseudomonas aeruginosa) recombinant protein POP, as well as preparation method and application thereof
  • PA (pseudomonas aeruginosa) recombinant protein POP, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction and Identification of Recombinant Plasmids

[0037] 1. The design sequence is the DNA sequence encoding the POP recombinant protein shown in SEQ ID NO: 1. The synthesis of the DNA sequence and the connection of the sequence and pGEX-6p-2 were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0038] 2. Transformation of recombinant plasmids

[0039] Transformation of recombinant plasmids 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 1ulPOP synthetic 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 place in a shaker at 37°C at 220rp for 1h.

[0040] Each tube was centrifuged at 5000 rpm for 3 min at room temp...

Embodiment 2

[0046] Example 2 Induced expression, purification and identification of expression form of recombinant fusion protein POP in prokaryotic expression system-Escherichia coli

[0047] 1. POP induced expression

[0048] Take 100 μL of the overnight cultured pGEX-6P-2-POP / 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 it to 20 mL of Amp+ resistant LB medium (The rest of the bacterial solution is stored in a refrigerator at 4°C for later use), culture at 37°C for 2-3 hours, rotate at 200 rpm, and reactivate until the OD600 is 0.8-1.0, add 4 μL of IPTG to make the final concentration 200 μM, and place on a shaker Induced expression Induced expression at 30°C for 3h.

[0049] 2) Take out the bacterial solution after induced expression, centrifuge at 12000rpm for 5min, discard the supernatant, add 1mL of lysisbuffer (20mMPB, pH7.2, 250mMNacl) to mix, u...

Embodiment 3

[0056] The preparation of embodiment 3POP antigen

[0057] 1. Amplify culture to obtain protein

[0058] Take 400 μL of the pGEX-6P-2-POP / XL-1blue bacterial solution stored in the refrigerator at 4°C and add it to 20mL LB medium containing Amp resistance for one activation. After incubating at 200rpm for 5-6 hours at 37°C, take 8mL once The activated bacterial solution was added to 400mL LB medium containing Amp resistance for secondary activation, cultured at 37°C for 3-4h until the OD600 was 1.0, then added 80μLPTG (final concentration 200μM) and placed in a shaker at 16°C overnight to induce Afterwards, 12000rpm centrifugal 15min collects thalline, adds 20mLlysisbuffer (same as embodiment 2) after resuspending thalline, bacterium liquid is carried out ultrasonic cracking 3min (200V), collect supernatant and GlutathioneSepharose4B (GE company) gel beads (beads) binding treatment; then SDS-PAGE gel electrophoresis.

[0059] 2. Use the enzyme digestion method to separate the...

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Abstract

The invention provides PA (pseudomonas aeruginosa) recombinant protein POP, as well as a preparation method and an application thereof. The recombinant protein is formed by sequential connection of a PA III type secretory system component protein PcrV fragment, a PA outer membrane lipoprotein OprI fragment and a PA IV type pilin PilA fragment through a linker. The recombinant protein has a general formula: PcrV fragment-(Linker A)m-Oprl fragment-(Linker B)n-PilA fragment, wherein sequences of the Linker A and the Linker B are respectively independently selected from one of GGGGS, GGSGG and YAPVDV; values allowed for m and n are 1, 2, 3 and 4 independently and respectively. The invention also provides the preparation method and the 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 drugs, preventive drugs or therapeutic drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmacy, and relates to a Pseudomonas aeruginosa recombinant protein POP. The invention further relates to a preparation method of the recombinant protein and its application in preparing vaccines and detection kits. Background technique [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA), commonly known as Pseudomonas aeruginosa, belongs to the genus Pseudomonas in non-fermenting bacteria. It is 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 respiratory tract, etc., and can also cause systemic infections such as endocard...

Claims

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Application Information

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