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Recombinant protein photosensitizer for targeting epidermal growth factor receptor (EGFR) and preparation method of recombinant protein photosensitizer

A recombinant protein and photosensitizer technology, applied in the field of recombinant protein photosensitizer preparation, can solve the problems of increased toxic and side reactions, high cost, low yield and the like, and achieves the effect of overcoming chemically coupled photosensitizers, low cost and high yield

Inactive Publication Date: 2014-05-14
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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  • Recombinant protein photosensitizer for targeting epidermal growth factor receptor (EGFR) and preparation method of recombinant protein photosensitizer
  • Recombinant protein photosensitizer for targeting epidermal growth factor receptor (EGFR) and preparation method of recombinant protein photosensitizer
  • Recombinant protein photosensitizer for targeting epidermal growth factor receptor (EGFR) and preparation method of recombinant protein photosensitizer

Examples

Experimental program
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Effect test

Embodiment 1

[0033](2) Fermentation and purification of recombinant protein photosensitizer: take 5ml of recombinant strains, inoculate with 100ml LB liquid medium, culture at 37°C for 8h, transfer to 28°C for culture, induce expression with IPTG for 12h, sonicate the cells, and use nickel affinity Chromatographic column purification of protein, SDS-PAGE gel electrophoresis detection of protein, such as figure 2 As shown, the protein size is about 22kD, and the 6×His tag is removed by TAGZyme to obtain a purified recombinant protein photosensitizer.

[0034] (3) Analysis of optical properties of recombinant proteins: Scan two optically active recombinant proteins at 400-800nm ​​with a visible spectrophotometer to observe the characteristic absorption peaks of the proteins; use a fluorescence spectrophotometer to analyze the fluorescence spectra of the two recombinant proteins Properties were detected, and the excitation wavelength and excitation peak of the protein were observed. The rec...

Embodiment 2

[0038] (2) Fermentation and purification of recombinant protein photosensitizer: take 800ml of recombinant strains, inoculate in a 5L fermenter containing 4L LB liquid medium, incubate at 37°C for 4h, add 800ml of feeding medium for another 3h, transfer to 28°C Cultivate, induce expression with IPTG for 12 hours, sonicate the bacteria, purify the protein with a nickel affinity column, and detect the protein by SDS-PAGE gel electrophoresis, such as figure 2 As shown, the protein size is about 22kD, and the 6×His tag is removed by TAGZyme to obtain a purified recombinant protein photosensitizer.

[0039] (3) Analysis of optical properties of recombinant proteins: Scan two optically active recombinant proteins at 400-800nm ​​with a visible spectrophotometer to observe the characteristic absorption peaks of the proteins; use a fluorescence spectrophotometer to analyze the fluorescence spectra of the two recombinant proteins Properties were detected, and the excitation wavelength ...

Embodiment 3

[0043] (2) Fermentation and purification of recombinant protein photosensitizer: take 800ml of recombinant strain, inoculate in a 5L fermenter containing 4L LB liquid medium, incubate at 37°C for 4h, inoculate into a culture tank containing 80L medium, incubate for 4h, Add 15L of feed medium, culture at 37°C for another 3h, transfer to 28°C for culture, induce expression with IPTG for 12h, sonicate the bacteria, purify the protein with a nickel affinity column, and detect the protein by SDS-PAGE gel electrophoresis, such as figure 2 As shown, the protein size is about 22kD, and the 6×His tag is removed by TAGZyme to obtain a purified recombinant protein photosensitizer.

[0044] (3) Analysis of optical properties of recombinant proteins: Scan two optically active recombinant proteins at 400-800nm ​​with a visible spectrophotometer to observe the characteristic absorption peaks of the proteins; use a fluorescence spectrophotometer to analyze the fluorescence spectra of the two ...

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Abstract

The invention discloses a recombinant protein photosensitizer for targeting an epidermal growth factor receptor and a preparation method of the recombinant protein photosensitizer, relating to the field of a genetic engineering technology and an antitumor biological medicine. A recombinant protein disclosed by the invention sequentially comprises an epidermal growth factor (EGF) interference sequence (20 amino acids of 1-20), a linker protein sequence (15 amino acid sequences of 21-35), a phycocyanin alpha sub-unit sequence (159 amino acid sequences of 36-194), and a 6*His-containing tag sequence (6 amino acid sequences of 195-200) from an end N to an end C. The recombinant protein is capable of directly targeting the tumor cell surface highly expressing the EGFR, and expressing a stronger effect of photoinduced-killing tumor cells through 630-nm laser irradiation for 20 minutes, and the inhibition rate reaches 95 percent; the recombinant protein is high in expression quantity, easy to purify, stable in performance, free from containing an organic solvent, and good in targeting, 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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Patent Type & Authority Applications(China)
IPC IPC(8): A61K41/00C07K19/00C12N15/62C12N15/70A61P35/00
Inventor 张伟杰王永刚马建忠冷非凡蒲秀瑛陈卓张庭瑞
Owner LANZHOU UNIVERSITY OF TECHNOLOGY
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