A kind of recombinant natriuretic peptide and its preparation method

A technology of natriuretic peptide and atrial natriuretic peptide, applied in botany equipment and methods, biochemical equipment and methods, chemical instruments and methods, etc., can solve the problem of short half-life, weak physiological effect, and acute heart failure treatment effect Unsatisfactory problems, to achieve the effect of prolonging the half-life of the drug, simple and feasible preparation process, and preventing and treating heart failure

Active Publication Date: 2021-02-19
SHENZHEN UNIV
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  • Claims
  • Application Information

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Problems solved by technology

[0011] The technical problem to be solved by the present invention is to provide a recombinant natriuretic peptide and its preparation method, which overcomes the problems caused by the very short half-life and weak physiological effect of the atrial natriuretic peptide in the prior art when treating acute heart failure. Deficiencies of unsatisfactory therapeutic effects in acute heart failure

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  • A kind of recombinant natriuretic peptide and its preparation method
  • A kind of recombinant natriuretic peptide and its preparation method
  • A kind of recombinant natriuretic peptide and its preparation method

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preparation example Construction

[0052] The present invention also provides a method for preparing the above-mentioned genetically engineered novel recombinant natriuretic peptide, the method comprising the following steps:

[0053] S1. Respectively obtain the N-terminal gene fragment of the atrial natriuretic peptide gene and the C-terminal gene fragment of the snake natriuretic peptide gene. Preferably, the obtained atrial natriuretic peptide gene is human atrial natriuretic peptide gene.

[0054] S2. Synthesize a recombinant natriuretic peptide gene by PCR, the N-terminal of the recombinant natriuretic peptide gene is the N-terminal gene fragment of the atrial natriuretic peptide gene, and the C-terminal of the recombinant natriuretic peptide gene is the C-terminal gene fragment of snake natriuretic peptide gene;

[0055] S3, constructing an expression vector;

[0056] S4. Expression.

[0057] Preferably, the above step S1 further includes:

[0058] S101. Obtaining the full-length human atrial natriure...

Embodiment 1

[0085] The novel recombinant natriuretic peptide (ADNP) gene that was amplified by PCR splicing and sequencing was cloned into the prokaryotic system Escherichia coli expression vector pGEX-4T-1, and a prokaryotic expression plasmid containing the novel recombinant natriuretic peptide (ADNP) was constructed pGEX-ADNP. BL21 and Rosetta were used as prokaryotic expression host bacteria. Extract high-quality and high-concentration prokaryotic expression plasmid pGEX-ADNP, using cold CaCl 2 Prepare competent host bacteria by conventional methods, transform the host bacteria by conventional methods, transform the above plasmids into BL21 and Rosetta strains respectively, and culture them at 37°C for 12-18 hours; pick a single clone expression strain, and inoculate LB medium (adding ampicillin , with a final concentration of 100 μg / ml), shake (250-300 rpm) at 37°C for overnight culture, collect the bacterial liquid by centrifugation, extract protein after ultrasonic crushing, and p...

Embodiment 2

[0087] The novel recombinant natriuretic peptide (ADNP) gene amplified by PCR splicing and sequencing was cloned into the prokaryotic expression vector pCW, and the prokaryotic expression plasmid pCW-ADNP containing the novel recombinant natriuretic peptide (ADNP) was constructed. BL21 and Rosetta were used as prokaryotic expression host bacteria. Extract high-quality and high-concentration prokaryotic expression plasmid pCW-ADNP, using cold CaCl 2 Competent host bacteria were prepared by conventional methods, and the above plasmids were transformed into BL21 and Rosetta strains respectively by conventional methods, and cultured at 37°C for 12-18 hours; single-clonal expression strains were picked and inoculated into LB medium (adding ampicillin, The final concentration is 100 μg / ml), cultured overnight at 37°C with shaking (250-300rpm), centrifuged to collect the bacterial liquid, extracted the protein after ultrasonic crushing, and carried out protein electrophoresis detecti...

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Abstract

The invention relates to a recombinant natriuretic peptide and a preparation method thereof. The recombinant natriuretic peptide is composed of 38 amino acids, and the sequence from the N-terminal to the C-terminal is: S L R R S S C F G G R M D R I G A Q S G L G C P S L R D P R P N A P S T S A wherein two cysteine ​​residues form a disulfide key ring structure. The novel recombinant natriuretic peptide of the present invention has the biological characteristics of atrial natriuretic peptide and snake natriuretic peptide at the same time, the physiological effect is significantly enhanced, and the half-life of the drug is obviously prolonged; It can effectively act on effector organs such as the heart, blood vessels and kidneys, exert diuresis, excrete sodium, increase glomerular filtration rate, expand blood vessels, etc., so as to effectively protect the physiological functions of the above organs, and achieve the prevention and treatment of serious heart failure and renal failure. disease.

Description

technical field [0001] The invention relates to the technical field of bioactive peptides, and more specifically, relates to a recombinant natriuretic peptide and a preparation method thereof. Background technique [0002] Heart failure is caused by abnormalities in myocardial function, structure, rhythm, conduction, etc., and eventually leads to serious heart diseases such as impaired ventricular pumping ability and filling function. Among them, congestive heart failure is a serious stage of heart failure, affecting 1-2 The health of the adult population, especially about 6-10% of the population over the age of 65, is threatened by this disease, becoming the most serious cardiovascular disease at present. [0003] Since Bold et al. discovered in 1981 that rat atrial extracts had natriuretic activity, members of the natriuretic peptide family have been discovered one after another. So far, a variety of natriuretic peptide molecules have been discovered, such as atrial natriu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/58C12N15/16C12N15/70
Inventor 李凌云周兆平王晓红章刚林枫
Owner SHENZHEN UNIV
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