Novel chimeric flagellin adjuvant of helicobacter pylori multi-epitope vaccine

A technology of Helicobacter pylori and flagellin, which is applied in the field of biomedicine, can solve the problems of stimulating mucosal immune response and recognition, etc.

Active Publication Date: 2014-06-18
CHINA PHARM UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the special structure of Hp flagellin, although it can trigger the body to produce a specific antibody response, it cannot be recognized by T...

Method used

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  • Novel chimeric flagellin adjuvant of helicobacter pylori multi-epitope vaccine
  • Novel chimeric flagellin adjuvant of helicobacter pylori multi-epitope vaccine
  • Novel chimeric flagellin adjuvant of helicobacter pylori multi-epitope vaccine

Examples

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example 1

[0030] Example 1: Molecular structure design of novel chimeric flagellin CF

[0031] Search the nucleotide sequence of D0 and D1 regions of Salmonella typhimurium flagellin Flic (GenBank: AEO27871.1) and the nucleotide sequence of D2 and D3 regions of Helicobacter pylori 26695 flagellin FlaA in the GenBank database, wherein Flic The nucleotide sequence corresponding to the D0 region is 1-426, the nucleotide sequence corresponding to the D1 region of Flic is 1255-1515, and the nucleotide sequence corresponding to the D2-D3 region of FlaA is 559-909.

[0032] According to the analysis of the bioinformatics software DNAstar, the spacer sequence (GGGGS) is easy to form turns and β-sheets, and is not easy to form α-helices. It is a flexible structure and can effectively space Flic / D0, FlaA / D2-D3 and Flic / D1. It is beneficial to the effective presentation of the Helicobacter pylori flagellin FlaA epitope and shows a high renaturation efficiency.

[0033] Results: The molecular struct...

example 2

[0034] Example 2: Construction of recombinant expression vector pETCF (containing chimeric gene CF)

[0035] The gene sequence of the designed chimeric flagellin CF was sent to Nanjing Sipujin Biological Co., Ltd. for codon optimization and whole gene synthesis, and cloned into pET28a.

[0036] Results: Supkin Biological Co., Ltd. used T7 promoter and terminator universal primers for gene sequencing, and proved that the nucleotide sequence of the chimeric gene CF in pETCF synthesized by PCR was completely correct, and there was no frameshift mutation. The plasmid map of the recombinant expression vector pETCF such as ( figure 2 ) shown.

example 3

[0037] Example 3: Prokaryotic Expression of Chimeric Flagellin

[0038] The recombinant plasmid pETCF synthesized by Sipujin Biological Co., Ltd. was transformed into E.coli BL21(DE3) strain. On the pre-prepared LB plate containing 50 μg / mL Kan, inoculate the loop-streaked genetically engineered strain BL21(DE3) / pETCF, place it upside down in a 37°C incubator, and after cultivating overnight for 12-16 hours, pick a single colony and inoculate it in In LB medium containing 50 μg / mL Kan, culture at 37°C, 180 rpm, for 12-16 hours. Sent to Sipujin Biological Co., Ltd. for sequence determination.

[0039] Inoculate the correctly sequenced bacterial species with 2% inoculum in LB medium containing 50 μg / mL Kan, shake overnight at 37°C and 180 rpm, and inoculate the bacteria solution with 1% inoculum in LB medium containing 50 μg / mL Kan the next morning. In the LB liquid medium, shake the flask at 37°C and 180rpm for 3h (OD value is 0.6-0.8), add IPTG to make the final concentratio...

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Abstract

The invention provides a novel chimeric flagellin adjuvant of a helicobacter pylori multi-epitope vaccine. An active ingredient of the novel chimeric flagellin adjuvant of the helicobacter pylori multi-epitope vaccine is a polypeptide chain which mainly consists of regions D0 and D1 of salmonella typhimurium flagellin Flic, and regions D2 and D3 of helicobacter pylori flagellin F1aA. A nucleotide sequence of the protein adjuvant is synthesized through a PCR (polymerase chain reaction) technology, expressed in escherichia coli by utilizing an expression vector, and the adjuvant protein is obtained after protein purification.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a design, construction and preparation method of a novel chimeric flagellin of a Helicobacter pylori (Hp) multi-epitope vaccine. Background technique [0002] Flagellin is the main component protein of bacterial flagella. According to the structure and function of the flagellin gene, flagellin can be divided into four regions, namely the highly conserved N-terminal and C-terminal (D0, D1 regions) and the highly variable middle part ( D2, D3 regions), these complex surface complexes are involved in bacterial motility and also in the induction of specific immune responses. The body's recognition of foreign antigenic substances such as pathogenic microorganisms is mediated by specific receptors on cells. It has been confirmed that this recognition is mainly realized through Toll-like receptors (Toll-like receptors, TLRs) family molecules on the cell surface. TLRs can recogni...

Claims

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Application Information

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IPC IPC(8): A61K39/39A61K39/02C12N15/70C12N5/10C12N1/21C12R1/01
Inventor 奚涛邢莹莹宋辉陆园园吕小波
Owner CHINA PHARM UNIV
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