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Targeting recombinant protein photosensitizer and preparation method thereof

A technology of recombinant protein and photosensitizer, which is applied in the field of preparation of recombinant protein photosensitizer, can solve the problems of low yield, increased toxic and side effects, and high cost, and achieve high yield, improved application effect, and low cost

Inactive Publication Date: 2014-05-07
LANZHOU UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently commonly used targeted photosensitizers are obtained by chemical coupling, but their disadvantages are low yield, high cost, low activity, and the possibility of introducing organic solvents to increase toxic and side effects.

Method used

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  • Targeting recombinant protein photosensitizer and preparation method thereof
  • Targeting recombinant protein photosensitizer and preparation method thereof
  • Targeting recombinant protein photosensitizer and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] (1) Construction of recombinant strains: design primers EGFRi1 and EGFRi2 (SEQ ID 3 and SEQ ID NO: 4), linker1 and linker2 (SEQ ID NO: 5 and SEQ ID NO: 6), and APCα1 and APCα2 (SEQ ID NO: 7 and SEQ ID NO: 8); The total DNA was extracted from algae PCC7120, and the allophycocyanin α subunit gene APCα was cloned by PCR; the EGFRi-linker1 nucleotide sequence was obtained by overlapping extension PCR amplification with EGFRi1 and EGFRi2 as primers; the linker1 and linker2 were used as primers Perform overlap extension PCR amplification to obtain the nucleotide sequence of linker2-APCα1; use EGFRi-linker1 and linker2-APCα1 as templates, and use EGFRi1 and linker2 as primers to perform PCR amplification to obtain the nucleotide sequence of EGFRi-linker-APCα1; EGFRi-linker-APCα1 and APCα were used as templates, and EGFRi1 and APCα2 were used as primers to obtain the complete fusion protein gene sequence; the fusion gene sequence was cloned into the expression vector pCDF- cpc...

Embodiment 2

[0037] (1) Construction of recombinant strains: design primers EGFRi1 and EGFRi2 (SEQ ID 3 and SEQ ID NO: 4), linker1 and linker2 (SEQ ID NO: 5 and SEQ ID NO: 6), and APCα1 and APCα2 (SEQ ID NO: 7 and SEQ ID NO: 8); The total DNA was extracted from algae PCC7120, and the allophycocyanin α subunit gene APCα was cloned by PCR; the EGFRi-linker1 nucleotide sequence was obtained by overlapping extension PCR amplification with EGFRi1 and EGFRi2 as primers; the linker1 and linker2 were used as primers Perform overlap extension PCR amplification to obtain the nucleotide sequence of linker2-APCα1; use EGFRi-linker1 and linker2-APCα1 as templates, and use EGFRi1 and linker2 as primers to perform PCR amplification to obtain the nucleotide sequence of EGFRi-linker-APCα1; EGFRi-linker-APCα1 and APCα were used as templates, and EGFRi1 and APCα2 were used as primers to obtain the complete fusion protein gene sequence; the fusion gene sequence was cloned into the expression vector pCDF- cpc...

Embodiment 3

[0042] (1) Construction of recombinant strains: design primers EGFRi1 and EGFRi2 (SEQ ID 3 and SEQ ID NO: 4), linker1 and linker2 (SEQ ID NO: 5 and SEQ ID NO: 6), and APCα1 and APCα2 (SEQ ID NO: 7 and SEQ ID NO: 8); The total DNA was extracted from algae PCC7120, and the allophycocyanin α subunit gene APCα was cloned by PCR; the EGFRi-linker1 nucleotide sequence was obtained by overlapping extension PCR amplification with EGFRi1 and EGFRi2 as primers; the linker1 and linker2 were used as primers Perform overlap extension PCR amplification to obtain the nucleotide sequence of linker2-APCα1; use EGFRi-linker1 and linker2-APCα1 as templates, and use EGFRi1 and linker2 as primers to perform PCR amplification to obtain the nucleotide sequence of EGFRi-linker-APCα1; EGFRi-linker-APCα1 and APCα were used as templates, and EGFRi1 and APCα2 were used as primers to obtain the complete fusion protein gene sequence; the fusion gene sequence was cloned into the expression vector pCDF- cpc...

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Abstract

The invention provides a targeting recombinant protein photosensitizer and a preparation method thereof. The recombinant protein comprises, from N-terminal to C-terminal, epidermal growth factor (EGF) interference sequence (20 amino acids in total from 1 to 20)-linker protein sequence (15 amino acids in total from 21 to 35)-allophycocyanin alpha-subunit sequence (158 amino acids in total from 36 to 193)-6*His tag sequence (6 amino acids in total from 194 to 199) in sequence. The protein can be directly targeted to surface of tumor cells with high-EGFR expression level, and the protein shows strong photoinduced tumor cell killing effect after laser radiation at 650 nm for 20 min, with inhibitory rate of up to 100%. The protein has high expression level, stable performance and good targeting property, is easy for purification, does not contain organic solvent, and is a brand new fusion protein photosensitizer.

Description

technical field [0001] The invention belongs to the field of recombinant protein in biotechnology, and in particular relates to a preparation method of a recombinant protein photosensitizer. Background technique [0002] The annual incidence of tumors in my country exceeds 2.6 million, and an average of one person dies of tumors every 2.5 minutes. Therefore, tumors have become a high incidence that seriously threatens human health. Photodynamic therapy is the latest development of modern gratuitous treatment. It is favored by clinical treatment for its safety, effectiveness, small side effects and relatively low cost. In 1996, the United States approved photodynamic therapy for clinical use, and China approved it in 2003. For clinical use. The key factor that determines the effect of photodynamic therapy is the photosensitizer. Since the photosensitizer is only relatively selectively absorbed by tumor tissue and is not specific, a small amount of photosensitizer will inevit...

Claims

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Application Information

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IPC IPC(8): A61K41/00C07K19/00C12N15/62C12N15/70A61P35/00
Inventor 王永刚张伟杰马建忠冷非凡蒲秀瑛陈卓张庭瑞
Owner LANZHOU UNIVERSITY OF TECHNOLOGY
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