B-cell epitope of human melanoma antigen MAGE-A3 and application thereof

A MAGE-A3, melanoma antigen technology, applied in the field of prevention, treatment and diagnosis of MAGE-A3 positive tumors, can solve the problem of weak immunogenicity

Inactive Publication Date: 2013-01-09
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Synthetic peptide vaccines have entered phase II clinical trials, but weak immunogenicity is a major disadvantage

Method used

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  • B-cell epitope of human melanoma antigen MAGE-A3 and application thereof
  • B-cell epitope of human melanoma antigen MAGE-A3 and application thereof
  • B-cell epitope of human melanoma antigen MAGE-A3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0090] The present invention also provides a kit (kit) for detecting tumors positive for MAGE-A3 antigen expression. The kit contains: a solid phase carrier coated with the B Cell epitope recombinant protein. The preparation method of the kit (kit) includes: (1) coating the B cell epitope recombinant protein on a solid phase carrier (such as an ELISA reaction plate), and obtaining the B cell epitope coated and (2) placing the solid phase carrier coated with the B cell epitope recombinant protein obtained in (1) in a kit, thereby obtaining a tumor positive for MAGE-A3 antigen expression medicine box. The kit may also include reagents (such as enzyme-linked immunosorbent reagents) for detecting antigen-antibody reactions, or reagents for gene amplification (such as PCR reagents) in appropriate containers, and / or also Instructions for use (book) are included.

[0091] Detection purpose

[0092] The B cell epitope recombinant protein of the present invention can be used to pre...

Embodiment 1

[0102] Example 1, Preparation and Identification of MAGE-A3 B Cell Epitope Recombinant Protein

[0103] 1. Design of MAGE-A3 B cell epitope recombinant protein gene

[0104] Obtain the MAGE-A3 protein gene sequence and amino acid sequence from the network resource database (Genbank, Swiss-Prot); use the online network resource (EXPASY) and biological software DNASTAR to analyze the antigenicity, surface accessibility and flexibility of the MAGE-A3 amino acid sequence and transmembrane region and other parameters were analyzed ( figure 1 ), supplemented by the analysis results of the secondary structure of the MAGE-A3 protein, analysis and comparison of various parameters to comprehensively judge the B cell dominant epitope of MAGE-A3. At the same time, use software such as SYFPEITHI to predict CTL epitopes such as HLA-A2 and Th epitopes of HLA-DR on MAGE-3. The polynomial coefficients of the candidate epitopes are respectively calculated and analyzed to finally determine the...

Embodiment 2

[0111] Example 2. Immunogenicity Study of MAGE-A3 B Cell Epitope Recombinant Protein

[0112] Female BALB / c mice aged 6-8 weeks were randomly divided into 5 groups, 9 mice in each group: Groups 1-3 were the MAGE-A3B cell epitope protein immunization group, group 4 was the carrier protein Trx-His control group group and PBS blank control group. B cell epitope protein and Freund's adjuvant (FCA) 1:1 (W / W) fully emulsified evenly, at 0, 2, and 4 weeks respectively, 50 μg of B cell epitope protein or empty carrier protein per mouse, back Mice were immunized subcutaneously. The humoral immune effect of the immunized mice was detected, that is, the titer and maintenance time of specific serum IgG.

[0113] At 0, 3, 5, 7, and 12 weeks, mice in each group were blooded by docking their tails to detect serum IgG antibody levels by ELISA.

[0114] 1. Determination results of serum-specific IgG antibodies of immunized mice

[0115] The three B cell epitope proteins of purified MAGE-A3...

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Abstract

The invention relates to a preparation method and application of a B-cell epitope recombinant protein of human melanoma antigen MAGE-A3 protein. The invention discloses three B-cell epitope-containing polypeptides obtained by screening based on the full-length MAGE-A3 protein. The invention further discloses coded amino acids and nucleotide sequences of B-cell epitope-containing polypeptides. The invention also discloses application of B-cell epitope-containing polypeptides on prevention, treatment and diagnosis of MAGE-A3-positive tumors. The B-cell epitope recombinant protein of MAGE-A3 protein is very strong in immunogenicity and antigenicity, and the application prospect is good.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals and diagnosis, more specifically, the invention relates to three B cell epitopes from the human melanoma antigen MAGE-A3 protein and its encoding nucleic acid, its preparation method and its application in prevention, treatment and diagnosis Application in MAGE-A3 positive tumors. Background technique [0002] Human melanoma antigen genes (MAGE) genes were first discovered in human melanoma cell lines. It is a large family including A, B, C, D, E and F six subfamilies. Among them, the tumor antigen encoded by MAGE-A3 is a cytoplasmic protein with a molecular weight of 48KDa, consisting of 314 amino acid residues. The MAGE-A3 gene is not only expressed in malignant tumor tissues mainly melanoma, but also expressed in a large proportion in various tumors of various tissue types, such as lung cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma, bone and flesh Tumors, leu...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N15/12C12N15/63C12N1/21C12N1/19C12N5/10C12P21/02A61K38/10A61K48/00A61P35/00G01N33/68C12Q1/68
Inventor 朱冠保张丽芳程骏朱珊丽刘纳新薛向阳沈贤陈俊
Owner WENZHOU MEDICAL UNIV
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