Targeted peptide NGR of CD13 (aminopeptidase N) and application thereof

A technology targeting peptides and fusion proteins, applied in the direction of peptides, etc., can solve the problem of not showing anti-tumor activity

Active Publication Date: 2013-09-11
优锐生物医药科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the early preclinical research process, it was found that NGR-hTNF showed good anti-tumor effect at a dose of 0.2ug / m2, while at the same dose, hTNF showed no anti-tumor activity

Method used

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  • Targeted peptide NGR of CD13 (aminopeptidase N) and application thereof
  • Targeted peptide NGR of CD13 (aminopeptidase N) and application thereof
  • Targeted peptide NGR of CD13 (aminopeptidase N) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Design and screen NGR peptides with good binding ability to CD13 by conventional histochemical staining methods:

[0030] (1) Artificial chemical synthesis of the following polypeptides labeled with biotin at the C-terminus (N-terminal → C-terminal sequence):

[0031] CNGRVSTNGRC (SEQ ID NO: 1), CNGRNGRC (SEQ ID NO: 4), CNGRVSTC (SEQ ID NO: 5), CNGRNGRNGRC (SEQ ID NO: 6), CNGRVSTNGRVSTNGRC (SEQ ID NO: 7);

[0032] (2) Screening by conventional histochemical staining method: the above polypeptides were diluted with PBS to 1000pM, 100pM, 10pM, 1pM and 0.1pM respectively, and each sample (polypeptide dilution) was compared with human hepatocellular carcinoma paraffin tissue sections (after routine pretreatment). Treatment) incubate at room temperature for 30 minutes; wash with PBS buffer (phosphate buffer); then add diluted HRP-labeled SA (SA-HRP) and incubate at room temperature for 15 minutes; wash with PBS buffer; Aniline) solution was incubated for 15 minutes; washed ...

Embodiment 2

[0040] Expression and detection of NGR-TNFα fusion protein in Escherichia coli:

[0041] (1) Construction of recombinant expression vector:

[0042] (2) Carry out according to conventional molecular cloning techniques. It consists of the CD13 targeting peptide NGR and TNFα. The full length of the NGR-TNFα gene provided by the present invention is 507, encoding 168 amino acids, and its gene sequence is shown in SEQ ID NO:2. The corresponding amino acid sequence is shown in SEQ ID NO:3.

[0043] (3) Induced expression of the NGR-TNFα fusion protein in Escherichia coli:

[0044] (4) A. chemical synthesis method to obtain the target gene;

[0045] B. Construction of recombinant expression vector: the expression plasmid pBV220 was double-digested with restriction endonucleases (same as the restriction site of the primer), and then ligated into vector pBV220 under the action of ligase after digestion. The connection reaction system is:

[0046]

[0047] C. Obtain the expressi...

Embodiment 3

[0051] Purification of the NGR-TNFα fusion protein:

[0052] According to the conventional method, the process is: cation exchange column chromatography; ammonium sulfate salting out; hydrophobic column chromatography; anion exchange column chromatography. The brief experimental steps are as follows:

[0053] A. Induced engineering bacteria expressing NGR-TNFα;

[0054] B. Ultrasonic crush the engineering bacteria obtained in step A in an ice bath, centrifuge, and take the supernatant after it precipitates;

[0055] C. Dilute the supernatant obtained in step B 10 times with 10 mMPB (pH 6.0), and purify the resulting dilution by cation exchange column chromatography;

[0056] D. Add the purified product obtained in step C to 50% ammonium sulfate for precipitation (4°C, overnight);

[0057] E. Centrifuge the solution obtained in step D, and take the precipitate, which is dissolved in 2M ammonium sulfate;

[0058] F. the ammonium sulfate solution obtained in step E is purifie...

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Abstract

The invention provides NGR targeted peptide of CD13 (aminopeptidase N) and application thereof. The NGR targeted peptide has the amino acid sequence of CNGRVSTNGRC. The invention also provides a fusion protein NGR-TNF (tumor necrosis factor) alpha. The NGR-TNF alpha is the fusion protein formed by the NGR targeted peptide and a TNF alpha. The targeted peptide NGR of the CD13 is designed and screened by a conventional histochemical staining method; and then the NGR peptide is fused with a human TNF alpha by a gene recombination method to obtain the fusion protein NGR-TNF alpha. Through a greatamount of experimental analysis, people find that by the NGR-TNF alpha, the growth of tumor cell can be inhibited by inhibiting the growth of neovascularization in tumor in a targeted mode and the effect of resisting the tumor is achieved.

Description

technical field [0001] The present invention provides a CD13 targeting peptide NGR, its preparation method and its application in the preparation of tumor targeting drugs or preparations for diagnosis or treatment; the present invention also provides a fusion protein NGR-TNFα, its preparation method and its preparation Application in diagnosis or treatment of tumor-targeted drugs or preparations. Background technique [0002] Malignant tumors seriously endanger human health. A research report published by the World Health Organization shows that at present, more than 11 million malignant tumors are diagnosed every year in the world, and more than 8 million people die of illness. It is predicted that by 2020, the incidence of malignant tumors will increase by 50%, and new malignant tumors will be added Cancer patients will reach 20 million per year. In my country, malignant tumors have become the number one cause of death among urban residents in recent years, with 2.2 mill...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06
Inventor 倪健陈建鹤
Owner 优锐生物医药科技(深圳)有限公司
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