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Novel gonadotropin releasing hormone oriented fusion protein mutant

A fusion protein and mutant technology, applied in the direction of peptide/protein components, hybrid peptides, drug combinations, etc., can solve problems such as drug failure, easy shedding of chemotherapeutic drugs, and damage to normal cells

Active Publication Date: 2013-07-31
BEIJING YIDE XINAO BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These technologies have certain limitations in the treatment of tumors. For example, gonadotropin-releasing hormone is coupled with chemotherapy drugs. Due to the problem of the coupling method, the chemotherapy drugs are easy to fall off, resulting in drug failure. Selective, attacking normal cells before the bulk drug reaches the tumor site

Method used

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  • Novel gonadotropin releasing hormone oriented fusion protein mutant
  • Novel gonadotropin releasing hormone oriented fusion protein mutant
  • Novel gonadotropin releasing hormone oriented fusion protein mutant

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-GnRH is obtained through the recombination technique known to those skilled in the art, and the sequence of the gene fragment inserted into the vector is determined. After verifying the correctness of its gene sequence, amplify and save it, cut out the target gene fragment from the vector, insert it into the expression vector, and name the recombinant plasmid pGnRH. Then use CaCl 2 Transform Escherichia coli BL21 by using the method, spread it on the surface of the screening plate medium containing antibiotics, select positive coloni...

example 2

[0024] Example 2 Expression of the protein of interest

[0025] 1) Shake flask expression: the genetically engineered bacteria pGnRH / BL21 prepared as above was cultured in LB medium overnight in a shake flask (37°C, 210rpm), 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 5 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 38% 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: 30 gr...

example 3

[0039] Example three purpose protein purification:

[0040] (1) Hydrophobic chromatography

[0041] Use hydrophobic chromatography medium (Octyl Sepharose 4 Fast Flow), balance solution A with 25mM Tris-HCl, pH8, 1M ammonium sulfate, after equilibrium, load the centrifuged supernatant of the lysate (supplement ammonium sulfate to 1M), and balance with A After 5 column volumes, linear gradient elution, 0-20 column volumes, 1-0.1M ammonium sulfate, the fusion protein peak was collected.

[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 sa...

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Abstract

The invention relates to a fusion protein mutant capable of directionally killing tumour cells, which consists of an orienting part and an effect part, wherein the orienting part consists of gonadotropin releasing hormone (GnRH), the effect part consists of cytotoxin, and the fusion protein mutant has the characteristics that the tumour cells excessively expressing GnRH receptors can be directionally reached and killed.

Description

1. Technical field [0001] The current traditional methods of treating cancer include surgery, radiotherapy, and chemotherapy. Among them, the use of surgery is a very direct and effective method, but it is actually restricted by various complicated factors such as the type of disease and the course of the disease. In many cases, it is difficult to implement. Radiotherapy and chemotherapy are the most common methods to treat cancer, but it is well known that their side effects are very large, and the effect is mostly unsatisfactory. Therefore, the medical field is trying to find a biological treatment method with small side effects and significant drug efficacy to replace radiotherapy and chemotherapy. The astonishing speed and unique curative effect of the treatment have aroused the widespread attention of researchers engaged in tumor treatment. [0002] The present invention relates to a fusion protein, which uses gonadotropin-releasing hormone (GnRH) as the guiding part, can...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/19C12N1/21C12N5/10A61K38/16A61K47/48A61P35/00
Inventor 刘波刘存莲武靖
Owner BEIJING YIDE XINAO BIOLOGICAL TECH
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