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Super antigen fusion protein for cancer therapy and its producing method

A fusion protein and superantigen technology, applied in the fields of peptide/protein components, animal/human proteins, chemical instruments and methods, etc., can solve the problems of antibody drug cycle and huge investment cost, save drug development costs and reduce medical costs. , the effect of reducing the dose

Inactive Publication Date: 2005-06-22
孙嘉琳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the research and development cycle and investment cost of antibody drugs are very huge.

Method used

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  • Super antigen fusion protein for cancer therapy and its producing method
  • Super antigen fusion protein for cancer therapy and its producing method
  • Super antigen fusion protein for cancer therapy and its producing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Isolation of superantigen SEA gene

[0048] According to the conventional molecular biology experiment method (T. Maniatis, et al, Molecular cloning, Alaboratory manual, Second edition, Cold spring harbor laboratory, 1989), the phenol / chloroform extraction method was used to extract Staphylococcus aureus (Staphylococcus aureus FRI337) According to the sequence of the superantigen SEA gene in the literature (MJ Betley and JJ Mekalanos, J. Bacteriol., 170, 34-41, 1988), the primers were designed: (1) the forward primer containing the restriction enzyme cut site of SrfI, 5'-GAGCCCGGGCAGCGAGAAAAGCGAAGAAATAAAT-3'; (2) Reverse primer containing NotI restriction site, 5'-GTGCGGCCGCACTTGTATATAAATATATATCAATATGCAT-3'. Use this primer to carry out the PCR amplification reaction of the SEA gene. The amount of template is 0.1 μl, and the cycle conditions of the PCR reaction are: 95°C for 30 seconds → 55°C for 30 seconds → 72°C for 120 seconds, a total of 30 cycles of reaction...

Embodiment 2

[0050] Example 2. Isolation of epidermal growth factor EGF gene

[0051] Design primers based on the reported epidermal growth factor EGF gene sequence (J. Smith, et al, Nucleic Acids Res., 10, 4467-4482, 1982): (1) Forward primer containing SrfI restriction enzyme cleavage site, 5'- GAGCCCGGGCAATTCCGATAGCGAGTGT-3'; (2) Reverse primer containing NotI restriction site, 5'-GTGCGGCCGCTCTAAGTTCCCACCATTT-3'. The EGF gene was isolated from the Human breast carcinoma cDNA gene library (Clontech) by PCR, which encodes a 53 amino acid polypeptide. The amount of template is 0.1 μl, and the cycle conditions of the PCR reaction are: 95°C for 30 seconds → 55°C for 30 seconds → 72°C for 30 seconds, a total of 30 cycles of reactions, and finally 72°C for 10 minutes. The length of the DNA fragment is approximately 170 bp.

[0052] The DNA product was subjected to low melting point gel electrophoresis, the DNA fragment was recovered, and the fragment was treated with SrfI and NotI restriction enzy...

Embodiment 3

[0053] Example 3. Isolation of vascular endothelial cell growth factor VEGF

[0054] From the reported vascular endothelial cell growth factor VEGF gene sequence (PJKeck, et al, Science, 246, 1309-1312, 1989; E. Tischer, et al, J. Biol. Chem., 266, 11947-11954, 1991) The primers for VEGF-121 were designed in: (1) forward primer containing SrfI restriction enzyme cut site, 5'-GAGCCCGGGCGCACCCATGGCAGAAGGAGGA-3'; (2) reverse primer containing NotI restriction enzyme cut site, 5'-GTGCGGCCGCCCGCCTCGGCTTGTCACATTTTTCTTGTCTTGCTCTATCTTTCTT-3 '. The VEGF-121 gene was isolated from the Human breast carcinoma cDNA library (Clontech) by PCR, which encodes a 121 amino acid polypeptide. The amount of template is 0.1 μl, and the cycle conditions of the PCR reaction are: 95°C for 30 seconds → 55°C for 30 seconds → 72°C for 50 seconds, a total of 30 cycles of reactions, and finally 72°C for 10 minutes. The length of the DNA fragment is approximately 370 bp.

[0055] The DNA product was subjected to ...

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Abstract

Disclosed is a super antigen fusion protein for cancer therapy and its producing method, wherein the invention provides a method for constructing fusion protein of cytokine and super antigen, and the method of expression and purification of the fusion protein in escherichia coli, the used material is epidermal growth factor EGF and vessel endothelial cell growth factor VEGF and staphylococcus aureus enterotoxin SEA, two fusion protein types of EGF-SEA and VEGF-SEA can be constructed, the fusion proteins can be used for treating cancer.

Description

[Technical Field] [0001] The present invention relates to a new type of superantigen fusion protein that can be used for gene recombination for anti-cancer therapy, and describes the polynucleotide and amino acid sequences encoding the fusion protein, as well as their expression, isolation and purification in E. coli. [Background technique] [0002] The current drug treatment for cancer diseases is mainly based on chemical drugs, which have large side effects. While chemical drugs kill cancer cells, they also harm normal cells. Chemical drugs lack specific effects on cancer cells. [0003] In order to solve the problem of drug specificity, antibodies are a very effective tool, a commonly used cancer cell-specific targeting vector, which can specifically act on cancer cells. The antibody itself can block cancer cells, and its Fc fragment can cause cytotoxicity. Antibodies can also be attached to a toxin protein to guide the toxin protein to kill cancer cells. [0004] Superantigen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/485C12N15/62
CPCC07K14/485C07K2319/01C12N15/62A61P35/00
Inventor 孙嘉琳
Owner 孙嘉琳
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