A group of gene recombinant human corticotropin precursors for improving serum glucocorticoid levels and preparation method

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

Active Publication Date: 2022-07-29
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A group of gene recombinant human corticotropin precursors for improving serum glucocorticoid levels and preparation method
  • A group of gene recombinant human corticotropin precursors for improving serum glucocorticoid levels and preparation method
  • A group of gene recombinant human corticotropin precursors for improving serum glucocorticoid levels and preparation method

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Experimental program
Comparison scheme
Effect test

specific Embodiment approach 1

[0056] Embodiment 1: This embodiment expresses, isolates and purifies the recombinant ProACTH113, ProACTH72, ProACTH80 and ProACTH141 fusion proteins, and performs the following steps:

[0057] 1. The ProACTH141CDS sequence was optimized and designed according to the codon bias in the prokaryotic expression system to be suitable for high-efficiency expression in the E. coli expression strain BL21-DE3. The fusion protein prokaryotic expression sequence NdeI-HisTag-EKst-ProACTH141-SalI with His tag and enterokinase cleavage site was constructed using NdeI and SalI as the restriction sites at the upstream and downstream ends of the CDS sequence of the fusion expression protein. This sequence will serve as a template for PCR amplification of the 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, design the upstream primers NdeI-HisTag-EKst-ProACTH1...

specific Embodiment approach 2

[0068] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the pET21a plasmid is not used, but pET30a is used as the expression vector.

specific Embodiment approach 3

[0069] Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that pET30a or pET21a plasmid is not used, but pET28a is used as the expression vector.

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Abstract

The present invention relates to four kinds of gene recombinant human corticotropin (ACTH) precursor proteins with the effect of increasing the level of glucocorticoid in serum: ProACTH113, ProACTH72, ProACTH80, ProACTH141, and a preparation method of the four precursor proteins. ProACTH113 contains the polypeptide chain of amino acids 105-217 of Pro-opiomelanocortin (POMC), ProACTH72 contains the polypeptide chain of amino acids 105-176 of POMC, ProACTH80 contains the polypeptide chain of amino acids 138-217 of POMC, ProACTH141 contains the polypeptide chain of amino acids 77-217 of POMC; the four ACTH precursor proteins all contain the amino acid sequence of amino acids 1 to 39 of ACTH polypeptide. To facilitate purification, the N-termini of four precursor proteins, such as ProACTH113, ProACTH72, ProACTH80, and ProACTH141, were linked to enterokinase substrate sequence (EKst), and enterokinase with a histone tag was used to excise the N-terminal of these four precursor proteins in vitro. The excess peptides attached to the ends generate free ProACTH113, ProACTH72, ProACTH80, and ProACTH141, and then use a nickel column to remove the enterokinase linked with histone tags.

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 their DNA and protein sequences, as well as a preparation process of the group of precursor proteins. Background technique [0002] Corticotropin (Corticotropin), also known as adrenocorticotropic hormone (ACTH), is a 39 amino acid polypeptide hormone secreted by basophils in the anterior pituitary gland. Biosynthesis and metabolism of fat and protein, regulate cardiovascular and cerebrovascular functions, improve resistance, protect and regenerate nerve damage, anti-inflammatory, immunosuppressive, anti-toxin, anti-shock, etc. ACTH is the only effective drug for the clinical treatment of convulsions in infants under two years of age. The incidence of the disease is 0.5% to 1%, 6 to 33% of infants with convulsions die before the age of 3, and 70 to 90% of children with convulsi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/695C12N15/70C07K19/00C12N15/62
Inventor 王大勇武新丽
Owner HAINAN UNIVERSITY
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