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Preparation method of recombinant human corticotropin releasing factor

An adrenal cortex and factor releasing technology, applied in the field of genetic engineering, can solve the problems of steric hindrance, incision, low endoproteinase cleavage efficiency, etc., and achieve the effects of low production cost, improved recovery rate, and improved druggability

Active Publication Date: 2013-09-18
重庆科润生物医药研发有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in actual operation, the fusion protein molecule is much larger than the short peptide to be expressed, which will form steric hindrance, often resulting in low efficiency of endoproteinase cleavage, or inability to cut at all

Method used

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  • Preparation method of recombinant human corticotropin releasing factor
  • Preparation method of recombinant human corticotropin releasing factor
  • Preparation method of recombinant human corticotropin releasing factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of engineering bacteria expressing rhCRF

[0042] 1. CRF gene design and synthesis

[0043] According to the human CRF amino acid sequence registered in GenBnak (accession number: CAA23834.1), the 41 amino acid gene coding sequence of the mature peptide was selected, and synonymous mutations were carried out taking into account codon bias and gene sequence optimization in E. coli. And at the 5' end of the gene, the nucleic acid coding sequence corresponding to the enterokinase recognition sequence (DDDDK) and the linking peptide (GGGGSGGGGSGGGGS) were sequentially connected, and at the same time, the KpnI recognition sequence GATTCC was introduced at the 5' end of the entire sequence, and the NotI recognition sequence GCGGCCGC was introduced at the 3' end. The designed gene sequence was subjected to whole gene synthesis (completed by Shanghai Sangong).

[0044] 2. Construction of pET32-CRF / BL21(DE3) engineering bacteria

[0045] 2.1 Construction...

Embodiment 2

[0056] Example 2 Fermentation of engineering bacteria pET32-CRF / BL21 (DE3)

[0057] Inoculate the seed solution cultivated overnight into a suitable medium (such as LB, TB or other suitable medium) in the fermenter for cultivation, and culture the bacteria to OD 600 For about 25, add such as IPTG induction 3-4 hours. Collect the bacteria by centrifugation, suspend the bacteria in a suitable buffer solution (25mm Tris-HCl, 150 mmNaCl, pH8.0), homogenize under high pressure to break the bacteria, centrifuge the broken solution, collect the supernatant, and discard the precipitate.

Embodiment 3

[0058] Example 3 rhCRF purification

[0059] The bacteriostasis supernatant was loaded on the Ni2+-Chelating Sepharose Fast Flow (GE Healthcare) chelation chromatography medium treated with 0.2M NiSO4 and equilibrated with 20mM Tris-HCl (pH8.0), and 100mM imidazole (containing 20mM Tris-HCl, pH8.0) was eluted; the collected target fusion protein was desalted, and 0.5U enterokinase (50mM Tris-HCl, 2mM CaCl2, 0.1% Tween-20, pH8.0) was added to each 1mg fusion protein in Digest the fusion protein for about 16 hours at 4°C.

[0060] The digested target protein is loaded on Ni2+-Chelating Sepharose Fast Flow, and the target protein and fusion tag are simultaneously hung on the column. However, the binding capacity of the target protein on the column is low, and the target protein is eluted with a low concentration of imidazole (50mM), and the peak of the eluted protein is collected. The SDS-PAGE detection molecular weight is about 4700Da, the purity is greater than 95%, and the H...

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Abstract

The invention relates to a preparation method of a recombinant human corticotropin releasing factor, characterized in that: by artificially synthesizing a gene sequence of human corticotropin releasing factor mature peptide, the gene sequence is inserted into an escherichia coli expression vector with a thioredoxin label, the target gent fuses with the thioredoxin 3'end, the central part of the vector contains an associated peptide and an enterokinase recognition site, and the expression vector is converted into appropriate escherichia coli to obtain genetic engineering bacteria, the genetic engineering bacteria is subject to optimized high-density fermentation and purification to prepare Trx-CRF fusion protein, then the Trx-CRF fusion protein is subject to enterokinase cutting, and then separation and purification are carried out to obtain the recombinant human corticotropin releasing factor. The prepared recombinant human corticotropin releasing factor has the advantages of high purity, good activity, and low production cost.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, more specifically, to a method for preparing a biologically active polypeptide of human adrenocorticotropin releasing factor by using gene recombination technology. Background technique [0002] Cerebral edema is a serious complication of intracranial tumors and their treatment, and often occurs with other brain diseases and injuries, and is one of the main causes of brain dysfunction. Many pathological processes such as hypoxia, trauma, infarction, inflammation and poisoning can be accompanied by cerebral edema. It can cause a range of physical and psychological symptoms, such as seizures, muscle weakness, lack of coordination, and double vision. The death rate of brain tumors in my country ranks 7th among malignant tumors, and there is an increasing trend year by year. Among them, the incidence rate of primary brain tumors is 1 / 10000, and there are more men than women. The inciden...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12R1/19
Inventor 张伟辛渝李树刚于廷和但国平龚会英柴新娟李晓丽曹莉君张勇
Owner 重庆科润生物医药研发有限公司
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