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Gastrin-releasing peptide (GRP) guided fusion protein

A fusion protein and sequence technology, applied in the direction of peptides, hybrid peptides, specific peptides, etc., can solve problems such as inability to effectively control tumors, inability to efficiently kill tumor cells, and easy shedding of chemotherapy drugs.

Inactive Publication Date: 2011-11-23
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] These technologies have certain limitations in the treatment of tumors. For example, GRP is coupled with chemotherapy drugs such as paclitaxel. Due to the problem of the coupling method, the chemotherapy drugs are easy to fall off, resulting in drug failure. , before the whole drug reaches the tumor site, it is easy to invade normal cells; secondly, GRP is connected to the single-chain antibody, because the effect of the single-chain antibody is not as strong as that of the whole antibody, so it only plays the role of inhibiting tumor cells, and because the tumor cells The rapidity of proliferation cannot effectively control the tumor; GRP is coupled with light labeling or metal chelating agents, which are currently mainly used for diagnosis, and cannot effectively kill tumor cells as a treatment

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0022] Construction of Example 1 Recombinant Fusion Protein Particle and Acquisition of Engineering Bacteria

[0023] Using the designed amino acid sequence of SEQ ID NO1 as a template, according to the central rule, select the codon preferred by E. coli to obtain the corresponding nucleic acid sequence of the fusion protein, and then use the whole gene synthesis technology to obtain the above target gene fragment. The recombinant gene vector pUC-GRP2 was obtained through the recombination technique known to those skilled in the art, and the sequence of the gene fragment inserted into the vector was determined. After verifying the correctness of its gene sequence, amplify and save, cut out the target gene fragment from the vector, insert it into the expression vector, and name the recombinant plasmid pGRP2. Then use the CaCl2 method to transform Escherichia coli BL21DE3, spread it on the surface of the screening plate medium containing antibiotics, select the positive colony a...

example 2

[0024] Example 2 Expression of the protein of interest

[0025] 1) Shake flask expression: the genetically engineered bacteria pGRP2 / BL21DE3 prepared as above were cultured overnight in LB medium in shake flasks (37°C, 200rpm), and then inoculated into LB medium at a volume ratio of 1:30, at 37°C After culturing for 3 hours, 0.1 mM IPTG was added for induction for 4 hours. The collected bacteria were analyzed by SDS-PAGE electrophoresis, and it was found that the fusion protein (43KD) was mainly expressed in soluble form, and the expression amount accounted for 40% of the total protein of the bacteria.

[0026] 2) Large-scale fermentation expression:

[0027] a. Medium composition:

[0028] a) Seed liquid medium (LB):

[0029] Tryptone: 10 g / L, yeast powder: 5 g / L, sodium chloride: 10 g / L.

[0030] b) Tank culture medium (15 liters):

[0031] Disodium hydrogen phosphate: 250 grams, potassium dihydrogen phosphate: 110 grams, sodium chloride: 13. grams, ammonium chloride: 3...

example 3

[0039] Example three purpose protein purification:

[0040] (1) Hydrophobic chromatography

[0041] Hydrophobic chromatography medium (Octyl Sepharose 4 Fast Flow) was used. Equilibrium solution A was 25mM Tris-HCl, pH8, 1M ammonium sulfate. After 1 column volume, linear gradient elution, 0-20 column volume, 1-0.1M ammonium sulfate, collect the fusion protein peak.

[0042] (2) Anion exchange chromatography

[0043] Use Source 30Q anion exchange chromatography medium, balance solution A is 25mM Tris-HCl, pH8, balance, the sample obtained in the previous step is diluted 4 times with water, load, balance, linear gradient elution, 0-20 column volumes, 0- 0.5M NaCl, collect the target fusion protein peak.

[0044] (3) Anion exchange chromatography

[0045] Use Q Sepharose High Performance chromatography medium, the balance solution is 20mM PB, pH7.4, the sample obtained in the previous step is diluted 3 times with water for loading, after equilibrium, use 20mM PB, pH7.4, 0-1M ...

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Abstract

The invention relates to a fusion protein which can directionally kill tumor cells. The fusion protein comprises a guiding part composed of gastrin-releasing peptide (GRP) and an effect part composed of cytotoxin, and has a characteristic of directionally reaching and killing tumor cells which overexpress GRP receptors.

Description

1. Technical field [0001] At present, there are many methods to treat cancer, including surgery, radiotherapy and chemotherapy. Surgery is a very effective method, but limited by the type of disease and the course of the disease, it cannot be applied in many cases. Radiotherapy and chemotherapy are the most common methods for treating cancer. , but its side effects are large and the effect is not satisfactory, so people are trying to find a biological treatment with less side effects and high efficiency to replace radiotherapy and chemotherapy. [0002] The invention relates to a fusion protein, which uses gastrin-releasing peptide as the guiding part of the fusion protein, can specifically combine with tumor cells overexpressing gastrin-releasing peptide receptor, and uses Pseudomonas exotoxin A Deletion of PE40, and its C-terminal 5 amino acid mutations to Lys-Asp-Glu-Leu, that is, PE40KDEL as the effector part of the fusion protein, specifically kills tumor cells. 2. Back...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K47/48A61K39/104A61P35/00
Inventor 李树民莘旭妮李咏梅
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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