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Mucosal immunoadjuvant inducing Th1 immune response, and application thereof

A mucosal immune adjuvant and immune response technology, applied in the direction of medical preparations containing active ingredients, the use of carriers to introduce foreign genetic material, hybrid peptides, etc., can solve the problem of poor immune protection, short duration of immune response, antigenic Weak immunogenicity and other issues, to achieve good social and economic benefits, good immune effect, and broad market prospects

Inactive Publication Date: 2013-07-24
PLA NAVY GENERAL HOSIPTAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the current adjuvant research, the immunogenicity of the antigen is generally weak, the duration of the immune response induced by the mucosal immune vaccine is short, and the immune protection effect is not good. The present invention provides a method to improve the intensity of the immune response and change the type of immune response A new type of mucosal immune adjuvant that induces IgG2a antibodies that depend on Th1-type immune responses (Th1-type immune responses play an important role in anti-infection and anti-tumor responses)

Method used

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  • Mucosal immunoadjuvant inducing Th1 immune response, and application thereof
  • Mucosal immunoadjuvant inducing Th1 immune response, and application thereof
  • Mucosal immunoadjuvant inducing Th1 immune response, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Determination of RTB and cell specific binding active site

[0073] 1. Use molecular simulation to build 3E1 light and heavy chain antibody spatial conformation

[0074] The amino acid sequence of the heavy chain of the 3E1 antibody with independent intellectual property rights is as follows: (see sequence 1 in the sequence table)

[0075] S G A E L M K P G A S V K I S C K A T G Y T F S S Y W I E W I K Q R P G H G L E W I G D I L P G SG S T N Y N E K F K G K A T F T A D T S S N T A Y M Q L S S L T S E D S A V Y Y C S R S F Y Y N Y D GA Y F A Y W G Q G T L V T V S A A K T T P P S V Y P L A P G S A A Q T N S M V T L G C L V K G Y F P EP V T V T W N S G S L S S G V H T F P A V L Q S D L Y T ​​L S S S V T V P S S T W P S E T V T C N V A H PA S S T K V D K K I V P R D C

[0076] Select a template protein with high homology (PDB library number: 2V7H), perform sequence alignment, and the result ( figure 1 .). The conformation of antibody heavy chain (VH+CH1) obtained by mole...

Embodiment 2

[0090] Example 2. Construction, expression, purification and identification of GFP-nRTB fusion protein prokaryotic expression vector

[0091] 1. Material

[0092] PCR kit, plasmid extraction kit, gel recovery kit, lysozyme Lysozyme, DNA and protein Marker are all products of Shanghai Shenggong; PET-30a(+) prokaryotic expression vector, restriction enzyme Nco I, EcoRV, T4 DNA ligase and pGEM-T Easy were purchased from Promega. The PCR primer synthesis and DNA sequencing were completed by Shanghai Boya Biotechnology Co., Ltd.

[0093] Two, method results

[0094] 1. Construction and design of prokaryotic expression vector ( Figure 7. Figure 8. )

[0095] Use PET-30a(+) prokaryotic expression vector. After analysis with BIOSUN software, the restriction sites Nco I and EcoRV were selected. The protein is expressed from ATG, and the C-terminal of the recombinant protein has a stop codon.

[0096] 2. Design PCR primers according to the target gene sequence.

[0097] The small peptide and ...

Embodiment 3

[0117] Example 3. Preliminary evaluation of mucosal immune effect of GFP-nRTB fusion protein

[0118] 1. Material

[0119] IgG, IgG1, IgG2a, IgG2b, and IgG3 monoclonal antibodies are all products of Sigma. HRP-labeled goat anti-mouse antibody (GAM-HRP) is produced by Boaosen Biotechnology Co., Ltd.

[0120] Two, method results

[0121] 1. Preliminary evaluation of mucosal immune effect of GFP-nRTB fusion protein

[0122] The four fusion proteins and 50μg of GFP purified under the tongue were administered sublingually under the 0, 14, and 28 immunization procedures, and blood was taken from the tail on the 7th day after the third immunization. The serum levels of GFP IgG and immunoglobulin were detected. The levels of IgG1, IgG2a, IgG2b, and IgG3 were tested by indirect ELISA after the serum was diluted 1:100 with PH7.4 phosphate buffer. The results showed that the levels of GFP IgG, IgG2b, and IgG3 in the serum after P1-GFP immunization were not significantly different from those of ...

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Abstract

The invention relates to a mucosal immunoadjuvant inducing Th1 immune response. A bioinformatics technology is applied in the invention, the spatial conformation of a ricin B-chain monoclonal anti-3E1 and Ricin-B compound which has obtained a Chinese invention patent is determined through the action of an antibody-antigen compound, and a molecular stimulation technology is utilized to reasonably determine four Ricin B identifying active regions of 3E1 and derive four nucleotide sequences. Four GFP-nRTB fusion proteins are obtained through a molecular cloning technology. Results of experiments that the four proteins are used to immunize BALB / C mice through a sublingual way three times show that a P1-GFP protein in the four proteins excites Th1 immune response, and a high-level IgG2a antibody is generated. The capability and the possible molecular mechanism of the targeting GFP mucosal immune response of the P1-GFP protein are discussed. The mucosal immunoadjuvant inducing Th1 immune response, which is screened through the above scheme, can be used for preparing vaccines, immunomodifiers or treatment medicines.

Description

Technical field [0001] The invention relates to a mucosal immune adjuvant capable of directly targeting antigens to antigen-presenting cells, and its application in preparing vaccines, immunomodulators or therapeutic drugs. Background technique [0002] Mucosa is an important physiological barrier to prevent the invasion of pathogenic microorganisms in the body's natural immune system. More than 95% of the body's infections occur in the mucosa or invade the body from the mucosa. The resulting mucosal damage and mucosal immune function disorders often become important incentives for autoimmune diseases or opportunistic infections. Therefore, prevention of mucosal infections A pivotal place in human health [1] . [0003] Mucosal immunity can induce the local mucosa to produce secretory IgA (sIgA), IgM and IgG and other protective antibodies, and induce other parts to produce sIgA and body fluid immune response; at the same time, mucosal immunity can also induce mucosal and systemic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K19/00C12N15/11C12N15/63A61K39/39
Inventor 郭建巍秦力维冯健男
Owner PLA NAVY GENERAL HOSIPTAL
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