recombinant human corticotropin (ACTH) precursor for improving serum glucocorticoid levels and preparation method

A technology of glucocorticoid and gene recombination, which is applied in the direction of adrenocorticotropic hormone, chemical instruments and methods, recombinant DNA technology, etc., can solve the problems of high production cost, transmission of animal viruses and mycoplasma, etc.

Active Publication Date: 2019-05-31
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, ACTH for domestic clinical application is mainly extracted from animal tissues, the production cost is relatively high, and there is a potential risk of spreading animal viruses and mycoplasma

Method used

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  • recombinant human corticotropin (ACTH) precursor for improving serum glucocorticoid levels and preparation method
  • recombinant human corticotropin (ACTH) precursor for improving serum glucocorticoid levels and preparation method
  • recombinant human corticotropin (ACTH) precursor for improving serum glucocorticoid levels and preparation method

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Experimental program
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specific Embodiment approach 1

[0056] Specific embodiment 1: In this embodiment, expression, separation and purification of gene recombinant ProACTH113, ProACTH72, ProACTH80 and ProACTH141 fusion proteins are carried out according to the following steps:

[0057] 1. The ProACTH141 CDS sequence was optimally designed according to the codon preference in the prokaryotic expression system, so as to be suitable for high-efficiency expression in E. coli expression strain BL21-DE3. Using NdeI and SalI as the restriction sites at the upstream and downstream ends of the CDS sequence of the fusion expression protein, the prokaryotic expression sequence NdeI-HisTag-EKst-ProACTH141-SalI of the fusion protein with His tag and enterokinase restriction site was constructed. This sequence will serve as a template for PCR amplification of ProACTH113, ProACTH72 and ProACTH80 sequences. Specific steps are as follows:

[0058] Primer design: Using the above optimized NdeI-HisTag-EKst-ProACTH141-SalI sequence as a template, d...

specific Embodiment approach 2

[0068] Embodiment 2: This embodiment differs from Embodiment 1 in that pET21a plasmid is not used, but pET30a is used as the expression vector.

specific Embodiment approach 3

[0069] Embodiment 3: This embodiment is different from Embodiment 1 or 2 in that pET30a or pET21a plasmids are not used, but pET28a is used as the expression vector.

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Abstract

The invention relates to four recombinant human corticotropin (ACTH) precursor proteins having an effect of increasing glucocorticoid levels in serum: ProACTH113, ProACTH72, ProACTH80, ProACTH141, anda method for preparing these four precursor proteins. ProACTH113 contains the 105th to 217th amino acid polypeptide chain of Pro-opiomelanocortin (POMC), ProACTH72 contains the 105th to 176th amino acid polypeptide chain of POMC, ProACTH80 contains the 138th and 217th amino acid polypeptide chains of POMC, and ProACTH141 contains the 77th to 217th amino acid polypeptide chains of POMC; all of thefour ACTH precursor proteins comprise amino acid sequences 1 to 39 of the ACTH polypeptide. For purification, the N-terminus of the four precursor proteins, ProACTH113, ProACTH72, ProACTH80, and ProACTH141, are linked to an enterokinase substrate sequence (EKst), and excess peptide sections connected with the N-terminus of the four precursor proteins are excised in vitro using histone-linked enterokinase. The free ProACTH113, ProACTH72, ProACTH80, and ProACTH141 are generated, and the histone-linked enterokinase is removed using a nickel column.

Description

technical field [0001] The invention belongs to the field of genetic engineering and protein engineering pharmacy, and specifically relates to a group of corticotropin precursor proteins (ProACTH) and the sequence of DNA and protein thereof, as well as the preparation process of the group of precursor proteins. Background technique [0002] Corticotropin, also known as adrenocorticotropic hormone (ACTH), is a 39-amino acid polypeptide hormone secreted by basophils in the anterior pituitary gland. It can regulate the synthesis and secretion of GC, regulate sugar, Biosynthesis and metabolism of fat and protein, regulation of cardiovascular and cerebrovascular functions, improvement of resistance, protection and regeneration of nerve damage, anti-inflammation, immunosuppression, anti-toxin, anti-shock, etc. ACTH is the only specific drug for the clinical treatment of infantile convulsions under the age of two. The incidence of the disease is 0.5% to 1%, 6-33% of infantile convu...

Claims

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Application Information

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IPC IPC(8): C07K14/695C12N15/70C07K19/00C12N15/62
Inventor 王大勇武新丽
Owner HAINAN UNIVERSITY
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