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Fusion protein of human interferon and targeting peptide, and preparation thereof

A technology of fusion protein and interferon, which is applied in the field of fusion protein of human interferon and targeting peptide and preparation thereof, can solve the problems of intolerance of patients, large side effects, lack of targeting, etc., achieves good effect and is easy to prepare , anti-tumor effect

Active Publication Date: 2013-02-13
哈药集团股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It solves the problem that human interferon binds to the interferon receptors of other tissue cells after it enters the blood circulation and has no targeting. It also solves the problem of large side effects caused by the large dose of interferon in clinical treatment of tumors, and patients cannot tolerate it. suffer from defects

Method used

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  • Fusion protein of human interferon and targeting peptide, and preparation thereof
  • Fusion protein of human interferon and targeting peptide, and preparation thereof
  • Fusion protein of human interferon and targeting peptide, and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Artificial synthesis of gene fragments:

[0075] According to the codon usage frequency table of Escherichia coli, the codons highly expressed in Escherichia coli were selected, the gene sequences of IFNα-2b and targeting peptide S1 were artificially designed, and the synthesis was entrusted to Shanghai Sangon Bioengineering Co., Ltd.

[0076] Obtaining fusion gene fragments:

[0077] The upstream and downstream primers of human IFNα-2b and the upstream and downstream primers of S1 with Nde I and BamH I restriction sites are designed in sections, and the primer sequences are as follows:

[0078] Primer sequence: shaded sites for restriction restriction.

[0079] IS 1up 1: CATATGTGTGACCTGCCGCAGACCCACTCTC

[0080] IS1 low1:

[0081] GGAAGTCACGGTGGCTGTGTTCTTTAGAACGCAGAGATTCCTGC

[0082] IS1 up2:

[0083] GGAATCTCTGCGTTCTAAAGAACACAGCCACCGTGACTTCCAGCCTGT TC

[0084] IS1 low2: GGATCCTTAGTCACCACGGTCACCACGCATACCACC

[0085] After using the PCR method to obtain these two ...

Embodiment 2

[0091] Take 1 pET-IS 1 For engineering bacteria, prepare grade 1 and grade 2 seeds, insert them into fermenters with 10% inoculum size, carry out large-scale cultivation, induce expression, and collect the bacteria (see Figure 7 ), using bacteriostasis solution (20mM Tris, 1mMgSO 4 , 50 μg / ml lysozyme) to suspend the cells, stir at room temperature for 60 min, and then use an ultrasonic cell disruptor to crush in an ice bath, and obtain inclusion bodies after centrifugation.

[0092] Use inclusion body washing solution (0.3% Triton X-100, 20mM Tris-HCl, 5mM EDTA, pH8.0) to wash it 2-3 times to ensure that it is cleaned, collect inclusion bodies after centrifugation, and use (30mM Tris- HCl, 10mM DTT, 6M guanidine hydrochloride or 8M urea) extracts were denatured and extracted at a volume of 1:20, and the supernatant was taken after centrifugation, and the refolding solution (20mM Tris-HCl, 0.3mM GSSG) was used at a volume of 1:20 80 volume for dilution and renaturation.

...

Embodiment 3

[0095] (1) Inhibition of neovascularization:

[0096] First, qualitative filter paper is used as the sample carrier, and the IS is cut into 5mm2 pieces of paper. 1 The fusion protein was diluted with normal saline, and the blank control was directly added with normal saline dropwise. The eggs were first sterilized with iodine, and then further wiped and sterilized with 75% ethanol. At the air chamber of the egg, carefully poke a small opening on the top of the egg embryo with tweezers, and then peel off the surrounding eggshell and shell membrane to make the opening about 1.5cm×1.5cm in size. Carefully use pointed surgical forceps to pierce the air cell membrane from the separation between the air cell and the yolk sac, gently remove the upper air cell membrane, and expose the underlying cAM membrane. The microcarriers were added to the part of the yolk sac membrane with fewer blood vessels, and the corresponding dose of medicament was dropped, then sealed with adhesive tape...

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Abstract

The present invention provides a fusion protein of human interferon and a targeting peptide, and preparation thereof. The fusion protein has a structure of IFN-M-S, wherein S represents a targeting peptide, M represents a linkage peptide, IFN represents interferon, an amino acid sequence of S can be any one selected from S1, S2, S3 and S4, an amino acid sequence of M contains 0-10 amino acid residues, preferably 4-6 amino acid residues, and IFN can be any one of I type interferon and II type interferon.

Description

Technical field: [0001] The invention relates to a fusion protein of human interferon and targeting peptide, construction of engineering bacteria and preparation of the fusion protein. Background technique: [0002] Interferon (Interferon, IFN) was discovered by British scientists Isaacs and Lindeman in 1957. IFN is a highly biologically active glycoprotein secreted by recipient cells after human and animal cells are infected by viruses, or nucleic acids, bacterial endotoxins, and mitogens. It is an important part of the body's defense system , with a molecular weight of 15,000-21,000 Daltons. [0003] When interferon was discovered, people thought that its antiviral activity was its only characteristic. With the deepening of research, interferon also showed strong antitumor and immunomodulatory activities. Therefore, the study of interferon has attracted more and more attention from people. According to the differences in their source, sequence, activity, etc., interfero...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12N1/19C12N5/10C07K1/18
Inventor 李郑武李会成朱红杰王冰陈玉军王莹徐岩张海涛安晓丽姜媛媛李国军高晶曹翊婕刘宇庭王丽娜黄宇红
Owner 哈药集团股份有限公司
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