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Improved flagellin mucosa adjuvant from non-pathopoiesia bacteria source and preparation method thereof

A technology of mucosal adjuvant and flagellin, applied in the direction of resisting vector-borne diseases, medical preparations containing active ingredients, and pharmaceutical formulas, can solve problems such as immune side reactions and inflammatory reactions, and reduce antigenicity and Risk, effect of maintaining adjuvant activity

Active Publication Date: 2011-04-06
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because it is derived from pathogenic bacteria, it may be potentially dangerous, and it can also cause inflammation and generate a large number of immune responses against flagellin itself, leading to possible tolerance and other possible immune side effects

Method used

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  • Improved flagellin mucosa adjuvant from non-pathopoiesia bacteria source and preparation method thereof
  • Improved flagellin mucosa adjuvant from non-pathopoiesia bacteria source and preparation method thereof
  • Improved flagellin mucosa adjuvant from non-pathopoiesia bacteria source and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Construction of clones of different segments of KF and expression of antigenic proteins thereof

[0045] Construction of cloned fragments of different segments of KF such as figure 1 As shown, among them, Picture 1-1 is a protocol for constructing clones of different segments of KF, including KF1, KF2, KF3, KFD1 and SF3. Among them, KFD1 is a fragment with the hypervariable region removed and inserted into the NheI restriction site, KF1 is the hypervariable region fragment of KF, KF2 is the central fragment of the KF hypervariable region, and KF3 is the NheI restriction site inserted in the hypervariable region of KFD1 The KF recovery clone formed after the point, SF3 is a fragment that replaces the hypervariable region with the SF hypervariable region and inserts the NheI restriction site.

[0046] KF refers to the flagellin of E. coli K12 strain MG1655 substrain (E. coli K12 strain MG1655 substrain flagellin), and its gene sequence number is ID-949101. ...

Embodiment 2

[0053] Example 2: Determination of major antigenic segments of KF

[0054] figure 2 It is the result of determining the main antigenic segment as its internal hypervariable region by enzyme-linked immunosorbent assay (ELISA). in, diagram 2-1 The similarities and differences of the proportions of antibodies against different segments of KF in the sera of mice immunized with different immunization strategies were compared. The results in this figure show that the proportions of antibodies against different segments of KF in sera of mice immunized with different immunization strategies were basically the same. Figure 2-2 The characteristics of the proportion of antibodies against different segments of KF in the sera of mice immunized by the immunization strategy were compared. The results in this figure show that the antigenicity of the restored clone KF3 is basically the same as that of KF. The antigenicity of the hypervariable region KF1 is ≈90%, the central part of the hy...

Embodiment 3

[0055] Example 3: KF structure modification scheme and identification of fragments obtained by modification

[0056] Construct the KFD fragment of the conserved region first, that is, on the basis of the KF fragment, remove the KF hypervariable region, connect the N-terminal (KFN) and C-terminal (KFC-terminal) of KF to obtain the pET28a-KFD fragment; then the p24 fragment Insertion into pET28a-KFD yielded pET28a-KFD-p24. Three groups of KFN and four groups of p24 were designed, and two groups of KFN and four groups of p24 were selected for combined transformation, and eight modified fragments were obtained. details as follows:

[0057] image 3 It is the identification result of the KF structural transformation scheme and the fragments obtained by the transformation. The KF fragment transformation scheme is as follows: Figure 3-1 As shown, wherein, the primers used for KFN fragment transformation are as follows:

[0058] KF N1 section

[0059] Upstream primer (NcoI)

[...

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Abstract

The invention provides an improved flagellin mucosa adjuvant from a non-pathopoiesia bacteria source and a preparation method thereof. The method comprises the steps of removing the main antigen active region of flagellin by using flagellin mucosa adjuvant, replacing the antigen to be a destination antigen and adding a flexible structure with low immunogenicity, wherein the gene sequence of the improved flagellin mucosa adjuvant obtained with the method is SEQ ID NO:1-11, and the amino acid sequence is SEQ ID NO:22-32. With respect to the adjuvant, the potential danger, the antigenicity, the immunogenicity and the inflammatory response and toxicity caused thereby are reduced, the antigen presentation effect is improved, and the similarity of a three-dimensional structure of the adjuvant and a natural flagellin protein is maintained by adding the flexible structure with low immunogenicity so as to maintain the activity of the adjuvant.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an optimized and transformed flagellin mucosal adjuvant derived from non-pathogenic bacteria and a preparation method thereof. Background technique [0002] It is now known that the flagellin protein derived from pathogenic bacteria has an immune adjuvant effect, which is through the combination of flagellin protein and Toll-like receptors (Toll-like receptors, TLRs) 5, triggering the NF-κB pathway to trigger innate immunity, This in turn triggers specific immunity. By immunizing after mixing or fusion of flagellin protein and target protein, the immune response to the target antigen can be significantly improved, and the effect of resisting pathogenic microorganisms with the target antigen can be achieved. However, because it is derived from pathogenic bacteria, it may be potentially dangerous, and it can also cause inflammation and generate a large number of immune responses again...

Claims

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Application Information

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IPC IPC(8): A61K39/39A61P37/04
CPCY02A50/30
Inventor 鄢慧民杨菁毅刘芳
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI