Multi-epitope fusion protein for preventing pseudomonas aeruginosa infection and encoding gene, expression vector and application thereof

A Pseudomonas aeruginosa and fusion protein technology, applied in the field of genetic engineering, can solve problems such as failure of vaccine development, achieve good immunogenicity and prevent infection

Active Publication Date: 2021-04-13
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional vaccines mainly rely on antibodies, but PA has a high degree of diversity and variability, which may be the main reason for the failure of the above-mentioned traditional vaccine development

Method used

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  • Multi-epitope fusion protein for preventing pseudomonas aeruginosa infection and encoding gene, expression vector and application thereof
  • Multi-epitope fusion protein for preventing pseudomonas aeruginosa infection and encoding gene, expression vector and application thereof
  • Multi-epitope fusion protein for preventing pseudomonas aeruginosa infection and encoding gene, expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Prediction analysis of antigenic protein dominant antigenic epitope

[0033] Neither the endpoints nor any values ​​of the ranges disclosed herein are limited to such precise ranges or values, and these ranges or values ​​are understood to include values ​​approaching these ranges or values. For numerical ranges, between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values ​​can be combined with each other to obtain one or more new numerical ranges, these values Ranges should be considered as specifically disclosed herein.

[0034] In a first aspect, the present invention provides a Pseudomonas aeruginosa vaccine recombinant protein, the vaccine recombinant protein is (a):

[0035] (a) a vaccine recombinant protein having the amino acid sequence shown in SEQ ID NO: 1;

[0036] According to the present invention, the recombinant protein of the Pseudomonas aeruginosa vaccine provided ...

Embodiment 2

[0079] Example 2 Identification of epitopes that induce Th17 responses in AmpC

[0080] 1. Synthesis of multiple candidate epitope peptides

[0081] Jiangsu GenScript Biotechnology Co., Ltd. was entrusted to synthesize the walking polypeptide according to the amino acid sequence of AmpC (NCBI Reference Sequence: NP_252799.1). The following is the corresponding amino acid sequence of the synthetic peptide, see Table 3:

[0082] Table 3: Amino acid composition of AmpC peptide library

[0083]

[0084]

[0085]

[0086] 2. AmpC-immunized mice and isolation of lung lymphocytes after immunization

[0087]AmpC protein solution with a concentration of 5 mg / ml and Curdlan stock solution with a concentration of 20 mg / ml were mixed in equal volumes for preparation. After the mice were anesthetized with isoflurane gas, the above suspension was inoculated intranasally, 10ul per mouse, and the inoculation time points were 0, 14, and 21 days respectively. Fourteen days after th...

Embodiment 3

[0090] Example 3 Construction, expression, purification and identification of PVAC recombinant expression vector

[0091] 1. Construction of the carrier

[0092] Commissioned Shanghai Sangon Bioengineering Co., Ltd. to synthesize DNA encoding PVAC (SEQ ID NO:2)

[0093] 2. Transformation of recombinant plasmids

[0094] Take 1 tube of Escherichia coli XL-1blue competent cells from the -80°C refrigerator, and add 1 μl of the synthetic pGEX-6P-PVAC plasmid. Ice bath for 50min, heat shock in 42℃ metal bath for 60s, rapid ice bath for 1min. Add 1000 μl LB blank medium, mix well, and place in a shaker at 37° C. for 1 hour at 220 rpm. Each tube was centrifuged at 5000 rpm for 3 min at room temperature, 900 μl of the supernatant was discarded, and the bacteria were resuspended, and 100 μl was spread on an Amp-resistant LB plate. Pick well-separated colonies on the transformation plate, inoculate them in Amp-resistant LB medium, and culture them overnight at 37°C with shaking.

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Abstract

The invention provides a fusion protein for preventing pseudomonas aeruginosa infection. The fusion protei comprises a first peptide fragment, a second peptide fragment and a third peptide fragment which are linearly connected and combined; the amino acid sequence of the first peptide fragment is SEQ ID NO:94; the amino acid sequence of the second peptide fragment is SEQ ID NO:95; and the amino acid sequence of the third peptide fragment is SEQ ID NO: 96. The invention further provides a recombinant gene for encoding the fusion protein and application of the fusion protein in preventing the pseudomonas aeruginosa infection. The fusion protein disclosed by the invention can effectively prevent infection of various serotypes of pseudomonas aeruginosa.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a fusion protein for preventing Pseudomonas aeruginosa infection, its encoding gene, expression vector and application. Background technique [0002] Pseudomonas aeruginosa (PA) is widely distributed in nature, normal human skin, intestinal tract, and respiratory tract, and is the most common opportunistic pathogen in clinical ventilator pneumonia (VAP). With the increasing drug resistance of PA, the effect of conventional antibiotic treatment is limited, and other means are urgently needed to prevent and control PA infection. Vaccines are the most effective means of preventing and controlling infectious diseases. Since the research and development in the 1970s, no PA vaccine has been successfully launched at home and abroad. There are four PA vaccines that have entered clinical trials, and three of them have failed . Traditional vaccines mainly rely on antibodies, but PA has...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21A61K39/104A61P31/04C12R1/19
CPCC07K14/21C12N15/70A61K39/104A61P31/04C07K2319/00C07K2319/21C07K2319/43C07K2319/20C07K2319/22C07K2319/41C07K2319/23C07K2319/40
Inventor 王颖顾江张怡程新高晨张卫军鲁东水曾浩邹全明
Owner CHENGDU OLYMVAX BIOPHARM
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