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VEGF-CRM197 recombinant fusion protein vaccine as well as preparation method and application thereof

A fusion protein, protein technology, applied in the field of biotechnology and medicine, can solve the problem of low immunogenicity of polypeptide vaccines

Active Publication Date: 2022-01-07
SHANGHAI HUIMMUTECH BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, peptide vaccines have the defect of low immunogenicity; on the other hand, whether VEGF peptide epitopes can stimulate sufficient anti-VEGF neutralizing antibodies has not yet been clinically confirmed

Method used

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  • VEGF-CRM197 recombinant fusion protein vaccine as well as preparation method and application thereof
  • VEGF-CRM197 recombinant fusion protein vaccine as well as preparation method and application thereof
  • VEGF-CRM197 recombinant fusion protein vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0205] Example 1: Screening for VEGF antigen fragments with low VEGF biological activity and high immunogenicity

[0206] VEGF121 (1-121), VEGF107 (1-107) and VEGF82 (24-105) were prepared by Escherichia coli expression system, and three VEGF fragments were detected by VEGF-responsive luciferase reporter cell lines after preparation.

[0207] The VEGF-responsive cell lines were plated in a 96-well plate. After the cells were fixed, three VEGF fragments were added respectively. The VEGF fragments were gradually diluted from 500ng / ml. After incubation for 24 hours, a luciferase substrate was added to detect the luminescence value.

[0208] like figure 1 The test results showed that higher concentration of VEGF121 could induce the expression of luciferase by binding to VEGFR2 on the cell membrane of the responding cell line through the signaling pathway, and the concentration and fluorescence value showed an S-shaped curve; neither VEGF107 nor VEGF82 at the highest concentration ...

Embodiment 2

[0212] Example 2: Construction and expression of pCDFDuet-1-(sumoVEGF107-CRM197)-DsbC plasmid

[0213] The DNA coding sequence of sumoVEGF107-CRM197 (as shown in SEQ ID NO: 10) was synthesized and constructed into the first open reading frame of the pCDFDuet-1 expression plasmid by means of gene synthesis, wherein the amino acid sequence encoding sumo is located in The N-terminal of VEGF107-CRM197 (SEQ ID NO: 1), the amino acid sequence of sumoVEGF107-CRM197 is shown in SEQ ID NO: 9; on the basis of the previous step, the DNA encoding E. coli disulfide bond isomerase DsbC The sequence (shown as SEQ ID NO: 11) was synthesized using gene synthesis means and constructed into the second open reading frame of the pCDFDuet-1 expression plasmid. After the identification, the construction of pCDFDuet-1-(sumoVEGF107-CRM197)-DsbC was completed.

[0214] Take 2 µL of the constructed pCDFDuet-1-(sumoVEGF107-CRM197)-DsbC plasmid and add it to BL21 (DE3) expression engineering bacteria, in...

Embodiment 3

[0216] Embodiment 3: Preparation of recombinant fusion protein VEGF107-CRM197

[0217] Take the expressed bacterium in Example 2, break the bacterium, and immediately use Ni affinity column chromatography on the supernatant, the washing condition is 10% Buffer B (containing about 50mM imidazole), and the elution condition is 50% BufferB ( Contains about 250mM imidazole). After the eluted product of affinity chromatography was cleaved by sumo tag-specific protease Ulp1, Ni sepharose FF affinity chromatography was used again to collect the fraction of the flow-through to remove the sumo tag still with the His tag and the uncleaved tag. Target protein, partial high-affinity label with Ni column.

[0218] Concentrate the VEGF107-CRM197 flow-through fraction without the tag after enzyme digestion, continue to use Sephacryl S200 molecular sieve chromatography to refine and purify, collect the peak of the target protein, and identify the protein sample by SDS-PAGE. Figure 4 As sho...

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Abstract

The invention provides a VEGF-CRM197 recombinant fusion protein vaccine as well as a preparation method and application thereof. Specifically, the invention provides a VEGF truncated body VEGF1-107 antigen fragment which loses VEGF biological activity but retains immunogenicity, and the VEGF truncated body VEGF1-107 antigen fragment is fused with a diphtheria toxin mutant CRM197 for recombinant expression to form a VEGF recombinant fusion protein. After the VEGF recombinant fusion protein is combined with a liquid adjuvant for use, high-titer and high-antibody generation in mice and rhesus monkeys can be induced, and combination of VEGF-A and a receptor thereof is blocked, so that the promotion effect of VEGF-A on vascular endothelial cell proliferation is inhibited.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and in particular relates to the preparation and application of a vascular endothelial growth factor (VEGF) vaccine. Background technique [0002] The VEGF / VEGFR signaling pathway is a crucial limiting step in tumor angiogenesis and a key factor in promoting tumor metastasis. Vascular endothelial growth factor A (VEGF-A) is the cytokine most closely related to the execution of tumor angiogenesis and lymphangiogenesis, and activates the VEGF / VEGFR signaling pathway, which can lead to epithelial cell survival, mitosis, metastasis and differentiation, and vascular penetration. It has been confirmed that VEGF-mediated high permeability of blood vessels is closely related to the metastasis of malignant tumors. Studies have shown that tumor tissue hypoxia leads to the gene expression level of VEGF-A, and the expression level of VEGF-A is significantly up-regulated in many tumor tissues, such...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/22C07K1/20C07K1/18C07K1/16C12N15/62A61K38/18A61K39/00A61P35/00A61P27/02
CPCC07K14/475A61K39/001135A61P35/00A61P27/02C07K2319/55A61K38/00
Inventor 张文耀陈国友
Owner SHANGHAI HUIMMUTECH BIOTECHNOLOGY CO LTD
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