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Polypeptide with binding affinity to human melanoma antigen A3 protein and use of polypeptide

A melanoma antigen and affinity technology, applied in the field of biomedicine, can solve problems such as bone marrow suppression

Active Publication Date: 2020-11-24
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Radioimmunotherapy with isotope-labeled antibodies can also lead to myelosuppression, etc.

Method used

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  • Polypeptide with binding affinity to human melanoma antigen A3 protein and use of polypeptide
  • Polypeptide with binding affinity to human melanoma antigen A3 protein and use of polypeptide
  • Polypeptide with binding affinity to human melanoma antigen A3 protein and use of polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0140] Example 1. Library Construction and Screening Research of MAGE-A3 Binding Polypeptides

[0141] A random combinatorial library of phage displaying MAGE-A3-binding polypeptides, that is, a library of many different SPA domain-related polypeptides, was constructed, and MAGE-A3-binding polypeptides were screened from the library, and their affinity was identified.

[0142] 1. Construction and identification of a random combinatorial phage display library of MAGE-A3 binding peptides

[0143] According to the amino acid sequence and structure of wild-type SPA-Z (Nilsson B et al., Protein Eng.1987; 1 (2): 107-113), random primers were designed for the coding sequences corresponding to its three helical structural regions, and amplified by PCR. A SPA coding sequence that can lead to random amino acid mutations was added and named SPA-N. According to the conventional method of molecular cloning, clone the SPA-N coding sequence into the pCANTAB5E vector through the Sfi I and No...

Embodiment 2

[0153] Example 2, MAGE-A3 binding polypeptide recombinant plasmid construction and prokaryotic protein expression and purification

[0154] 2 clones with higher ELISA reads were selected as before ( figure 1 Z in MAGE-A3 172. Z MAGE-A3 770), and Zwt as a negative control for MAGE-A3 binding polypeptides. In order to perform functional testing on the screened affibody molecules, construct recombinant plasmids, express and identify prokaryotic proteins, and prepare purified proteins.

[0155] 1. Construction and identification of recombinant plasmid of pET21a(+) / affibody

[0156] Design PCR primers and upstream primers with reference to the affibody gene sequence (GenBank: GY324633.1) Italics and underline indicate Ned I restriction site), downstream primers The italics and underline indicate the XhoI restriction site); the sequenced correct quaternary library monoclonal affibody Z was screened MAGE-A3 172. Z MAGE-A3 770 as a template, the affibody target gene (SEQ ID...

Embodiment 3

[0159] Example 3, Z MAGE-A3 Binding of affibody polypeptide to MAGE-A3 recombinant protein

[0160] To identify Z MAGE-A3The binding specificity of affibody polypeptides to MAGE-A3 protein, using surface plasmon resonance (SPR) to analyze and screen Z MAGE-A3 172. Z MAGE-A3 Specific binding of the 770 affibody molecule and its control Zwt affibody to the target protein MAGE-A3.

[0161] 1. Preparation of MAGE-A3 protein

[0162] The pET21a(+) / MAGE-A3 recombinant plasmid constructed and preserved in the laboratory was transformed into Escherichia coli BL21(DE3), and the recombinant protein was expressed after being induced by IPTG. The prepared protein was purified by Ni-NTA affinity chromatography, and routinely immunized Serum antibodies were prepared from Japanese white rabbits. As a result, SDS-PAGE electrophoresis showed that an obvious protein band appeared at the position of about 48kDa relative molecular mass (Mr), which was consistent with the expected protein Mr ...

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Abstract

The invention relates to a polypeptide with binding affinity to human melanoma antigen MAGE-A3 and use of the polypeptide. The polypeptide with binding affinity to the MAGE-A3 protein is disclosed forthe first time; and the invention also provides application of the polypeptide in diagnosis and detection, and the use of the polypeptide as a targeting carrier in the diagnosis or treatment of drugsor molecular targeting reagents.

Description

technical field [0001] The invention relates to the field of biomedicine, more specifically, the invention relates to a polypeptide with binding affinity to human melanoma antigen A3 protein (MAGE-A3) and its application. Background technique [0002] Tumor is a frequently-occurring and common disease in the world, and the mortality rate of malignant tumors ranks first among all diseases. Among them, the incidence and mortality of lung cancer rank first in the world, and gastric cancer ranks second in the death rate of malignant tumors. , breast cancer is the first cause of death in women. At present, the incidence of lung cancer in my country has shown an explosive growth trend, while the annual incidence of gastric cancer continues to rise, and shows a significant trend of younger age. The MAGE gene was first discovered in melanoma cell lines. It is a large family consisting of six subfamilies including A, B, C, D, E and F. The MAGE-A3 gene is not only expressed in malign...

Claims

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Application Information

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IPC IPC(8): C07K14/31C12N15/31A61K47/64A61K47/65A61P35/00A61P35/02G01N33/574
CPCC07K14/31A61K47/64A61K47/6415A61K47/65A61P35/00A61P35/02G01N33/5743G01N33/57492G01N2333/31
Inventor 张丽芳朱珊丽薛向阳蒋朋飞陈俊
Owner WENZHOU MEDICAL UNIV
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