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Vaccine universal vectors and preparation method and application thereof

A vaccine carrier and vaccine technology, applied in the field of vaccine universal carrier protein and its preparation, can solve the problems of weak particle uniformity, time-consuming, manpower and material resources, and affecting immunogenicity

Active Publication Date: 2020-12-11
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, inserted foreign sequences may lead to structural instability of VLPs, affecting assembly and immunogenicity
In addition, fusion expression to construct different recombinant proteins requires a lot of time, manpower and material resources
The chemical conjugation method requires a large amount of coupling reagents, and the conjugation sites on the surface of VLPs are uncertain, resulting in weak particle uniformity and affecting immunogenicity, and both fusion expression and chemical conjugation are not conducive to multi-target and personalized vaccines research and application

Method used

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  • Vaccine universal vectors and preparation method and application thereof
  • Vaccine universal vectors and preparation method and application thereof
  • Vaccine universal vectors and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1HB

[0123] Example 1HBc-S and HBc (1-183) Preparation of -S vaccine carrier protein

[0124] The amino-terminal truncated ΔN1SpyCatcher gene fusion was inserted between the 78th and 79th amino acids of the main immunodominant region of the truncated HBc (1-149), constructed as follows Figure 1A The shown vaccine carrier protein HBc-S; the coding gene fusion of amino terminal truncated ΔN1SpyCatcher is inserted between the 78th and 79th amino acids of the main immunodominant region of full-length HBc (1-183), constructed as Figure 1B The indicated vaccine carrier protein HBc (1-183) -S; In order to ensure the correct self-assembly of HBc and the correct display of SpyCatcher on the surface of HBc VLPs, introduce GSG linker and two glutamic acid (E) on both sides of the insertion site of ΔN1SpyCatcher gene, HBc-S-pBR and HBc (1-183) -S-pBR was constructed by Sangon Bioengineering (Shanghai).

[0125] HBc-S-pBR or HBc (1-183) -S-pBR was transformed into BL21 competent cells, 3 t...

Embodiment 2

[0127] Example 2 Application of HBc-S to prepare chimeric virus-like particle vaccines displaying linear, circular and modified epitopes

[0128] (1) Assembly characteristics of HBc-S

[0129] Concentrate the purified HBc-S to 3 mg / mL, and wash with 20 mM PBS (pH=5), 20 mM PBS (pH=6.2), 20 mM PBS (pH=8) and citric acid reaction buffer (Citrate Buffer, pH=6.2 ) diluted to 0.2mg / mL, add Protein gel stain (250×diluted); truncated HBc (1-149) was used as a control, heated from 20°C to 95°C using a real-time fluorescent quantitative PCR instrument, and the heating rate was 1°C / min.

[0130] The result is as Figure 4A , Figure 4B , Figure 4C , Figure 4D with Figure 4E As shown, the truncated HBc VLPs only had a Tm value of 83°C, indicating the existence of the VLP structure; while HBc-S only showed a Tm value of 83°C in the citric acid reaction buffer, indicating that HBc-S was in the citric acid solution Stable assembly into VLPs.

[0131] (2) Preparation of HBc-S-P V...

Embodiment 3

[0142] Example 3 Application of HBc-S-pTau422 Vaccine to Treat Tau.P301S Transgenic Mice

[0143] (1) Preparation of HBc-S-pTau422 vaccine

[0144]Mix HBc-S VLPs and SpTau422 at a molar ratio of 1:3, add 3 times the volume of citrate binding reaction buffer, and leave it at room temperature for 1-3 hours; then the mixture is ultrafiltered with a 100kDa cut-off membrane, Unreacted peptide SpTau422 was removed (citric acid reaction buffer as ultrafiltration liquid).

[0145] Figure 11A It is the result of SDS-PAGE detection, the electrophoretic band of HBc-S-pTau422 moves up, slightly larger than that of HBc-S, and the purity is higher, indicating that the SpTau422 polypeptide has undergone an adhesion reaction with HBc-S, realizing the full integration of HBc-S modification; Figure 11B It is the transmission electron microscope result picture, HBc-S-pTau422 maintains the intact VLP structure; Figure 11C Shown as a particle size distribution graph, the particle size of th...

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Abstract

The invention provides vaccine universal vectors HBc-S and HBc (1-183) -S which are constructed based on SpyCatcher / SpyTag and take hepatitis B virus core protein as a basis. SpyCatcher is displayed in a main immunodominance region of VLPs and can be subjected to an adhesion reaction with SpyTag at the amino terminal of epitope polypeptide, linear, annular and phosphorylated B cell epitopes SP aredisplayed on the surfaces of VLPs, and a series of HBc-S-P VLPs vaccines are prepared. Mice immunized with the vaccines can generate antibodies specific to the epitopes; and HBc-S-pTau 422 remarkablyimproves cognition and pathological changes of Alzheimer' S disease transgenic mice; HBc (1-183) -S induces Th1 type immunoreaction, and the HBc (1-183) -S-OVA vaccines are of a VLP structure, can activate and promote maturation of DCs, remarkably inhibit growth of tumors and prolong the lifetime of the tumors.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a universal vaccine carrier and its preparation method and application, in particular to a vaccine universal carrier protein fused and expressed by SpyCatcher protein and truncated hepatitis B virus core protein or full-length hepatitis B virus core protein, and its preparation method and application. Background technique [0002] Hepatitis B virus core protein is the nucleocapsid structural protein of hepatitis B virus (Hepatitis B), which can self-assemble into virus-like particles (Virus-like particle, VLP) with a particle size of about 30nm, which can easily enter lymph nodes. HBc VLPs particles are moderate in size, easy to be absorbed non-specifically by antigen-presenting cells in the way of endocytosis or pinocytosis, have a strong adjuvant effect, can activate the immune system in a T-cell-dependent or T-cell-independent manner, and are rich in T helper (Thelper, Th) cell epit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/36C07K1/30C07K1/18C07K1/16C12N15/62C12N15/70A61K39/385A61K39/39A61K39/00A61P25/28A61P35/00
CPCC07K14/005C12N15/70A61K39/385A61K39/39A61K39/0011A61K39/00A61P25/28A61P35/00C12N2730/10122C12N2730/10143C07K2319/00A61K2039/55505A61K2039/6075A61K2039/5258
Inventor 刘瑞田季梅谢喜秀
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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