Fusion protein modified by klebsiella pneumonia polysaccharide, and application of fusion protein

A technology for Klebsiella and pneumonia, applied in the field of biomedicine, can solve the problems of difficult vaccines, high cost, and few applications in the field of bacteria, and achieve the effect of improving immune effect and strong operability

Active Publication Date: 2020-11-10
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The O antigen, especially the O2 antigen, has low immunogenicity. As a vaccine alone, although the safety is well guaranteed, it cannot effectively stimulate the body's immune response, and often requires some adjuvants and suitable delivery systems. to promote the greatest immune response
In recent years, studies have found that nanoscale vaccines often have great application potential and play an important role in enhancing the immunity of weak antigens. Currently, they are widely used in tumor and virus vaccines, but less in the field of bacteria.
[0004] The preparation method of polysaccharide-conjugated vaccines currently on the market is a chemical method. The polysaccharide and protein are activated separately, and then cross-linked by chemical methods.
Since the preparation of the vaccine is completed through chemical reactions, monomeric proteins are often used, and it is difficult to make the vaccine reach the nanoscale

Method used

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  • Fusion protein modified by klebsiella pneumonia polysaccharide, and application of fusion protein
  • Fusion protein modified by klebsiella pneumonia polysaccharide, and application of fusion protein
  • Fusion protein modified by klebsiella pneumonia polysaccharide, and application of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1, Klebsiella pneumoniae with lipopolysaccharide synthesis deficiency

[0076] 1. Preparation of Klebsiella pneumoniae 355 strains (also known as KP355 or 355) with deletion of O-antigen ligase (waaL) gene

[0077] 1. Preparation of linear targeting DNA fragments

[0078] 1) Design of PCR primers

[0079] Compare the waaL gene of Klebsiella pneumoniae on NCBI with the local Blast database of the whole genome of 355 strains of Klebsiella pneumoniae, search for the waaL homologous gene on the genome of 355 strains, and then based on the gene and its upstream and downstream sequences , using Primer 6.0 software to complete the design of relevant primers (see Table 1 for details). A pair of primers, waaL-up-F / waaL-up-R and waaL-dw-F / waaL-dw-R, were designed respectively at the upstream (5' end) and downstream (3' end) of the waaL gene. For the convenience of operation, restriction enzyme cutting sites BamHI and SalI will be added to the end of the primer of the u...

Embodiment 2

[0110] Preparation of Example 2 Nano Glycoprotein Particles CTBTri4573-OPS

[0111] 1) Construction of the expression vector of the glycosylated fusion protein CTBTri4573

[0112] Construction of Neisseria meningitidis glycosyltransferase PglL expression vector: The amino acid sequence of Neisseria meningitidis glycosyltransferase PglL (GeneBank: JN200826.1) is shown in SEQ ID No.1, and its coding sequence is shown in SEQ ID No. 180-1994 nucleotides of .2. The 1st-6th nucleotide of SEQ ID No.2 is the XbaI recognition site, and the 105th-2240th nucleotide of SEQ IDNo.2 is the sequence of the PglL expression cassette. In the PglL expression cassette, the expression of PglL is controlled by tac The promoter is activated, and the expression cassette is named tacpglL. Wherein, the 105th-133rd nucleotide of SEQ ID No.2 is the sequence of the tac promoter, the 180th-1994th nucleotide is the coding sequence of Neisseria meningitidis glycosyltransferase PglL, and the 2475-2480th The...

Embodiment 3

[0130] The preparation of embodiment 3CTB4573-OPS

[0131] 1) Construction of the expression vector of the glycosylated carrier protein CTB4573

[0132] The encoded amino acid sequence of CTB4573 is shown in SEQ ID No.5, the gene sequence is shown in 178-654 of SEQ ID No.6, and the 1-6 nucleotides of SEQ ID No.6 are XbaI recognition sites, The 103-131 nucleotides are the sequence of the tac promoter, the 178-630 nucleotides are the coding sequence of CTB4573, the 631-636 nucleotides are the XhoI recognition sequence, and the 637-654 nucleotides are His tag. In the sequence, the CTB4573 sequence is a glycosylation modification sequence, and it is a peptide segment consisting of 29 amino acids at positions 45-73 of pili protein PilE.

[0133] Digest the DNA molecule shown in SEQ ID No.6 with XbaI and XhoI to obtain a gene fragment; digest pET28a(+) with XbaI and XhoI to obtain a large vector fragment; connect the gene fragment to the large vector fragment to obtain a recombina...

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Abstract

The invention discloses a fusion protein modified by klebsiella pneumonia polysaccharide, and application of the fusion protein. A preparation method for the fusion protein modified by the klebsiellapneumonia polysaccharide comprises the step of expressing the encoding gene of the protein in creatures to obtain the protein modified by the klebsiella pneumonia polysaccharide. The protein is a) orb) or c) or d), wherein a) is the protein formed by an amino acid sequence of SEQ ID No.3, b is the protein formed by an amino acid sequence of SEQ ID No.5, c) is the fusion protein obtained by carrying out protein tag fusion on the carboxyl terminal or / and amino terminal of the protein disclosed by a) or b), and d) is the protein obtained by carrying out substitution and / or deletion and / or addition on the amino acid sequence disclosed by a) or b) through one or multiple amino acid residues.

Description

technical field [0001] The invention relates to a polysaccharide-modified fusion protein of Klebsiella pneumoniae and an application thereof, belonging to the field of biomedicine. Background technique [0002] Klebsiella pneumoniae is a Gram-negative bacterium that is ubiquitous in the environment, on human and animal skin surfaces and mucous membranes. It is an opportunistic pathogen that can cause diseases such as pneumonia, endophthalmitis, bacteremia, and liver abscess. It is easy to infect young children and the elderly and infirm. It has become the most important pathogenic factor of hospital and community-acquired infections. Klebsiella pneumoniae is often drug-resistant, posing enormous difficulties in the treatment of its infection. In addition, the reported cases of Klebsiella pneumoniae capable of producing extended-spectrum β-lactamases have gradually increased in recent years, and clinically there is almost no drug available; in addition, the emergence of high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/11C12N1/21C12N5/10A61K39/108A61P31/04C12R1/22
CPCC07K14/00C12N15/74A61K39/0266A61P31/04C07K2319/00
Inventor 王恒樑朱力张璐璐潘超吴军孙鹏冯尔玲刘波曾明王斌
Owner ACADEMY OF MILITARY MEDICAL SCI
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