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Novel natriuretic peptide, preparation method and applications thereof in preparation of genetic engineering drugs

A new type of genetic engineering technology, applied in the field of biopharmaceuticals, can solve the problems of limited specific binding effect, short half-life of drugs, unsatisfactory prevention and treatment of acute heart failure, etc., to prevent and treat debilitating diseases, increase the intensity and duration of action, Increase the effect of the stimulating effect

Active Publication Date: 2014-02-26
SHENZHEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is that, when brain natriuretic peptide is used in the treatment of acute heart failure in the prior art, the effect on the prevention and treatment of acute heart failure is not good due to its limited site-specific binding effect and short half-life of the drug. The ideal defect is to provide a new type of natriuretic peptide prepared based on genetic engineering technology. This new type of natriuretic peptide has the characteristics of brain natriuretic peptide and snake natriuretic peptide. The specific effect is significantly enhanced, and the half-life of the drug is significantly prolonged. It can effectively act on the heart , blood vessels and kidneys and other effector organs, and effectively protect the functions of the heart, kidneys and other organs, to achieve the purpose of protecting, preventing and treating heart, kidney and other organ failure diseases

Method used

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  • Novel natriuretic peptide, preparation method and applications thereof in preparation of genetic engineering drugs
  • Novel natriuretic peptide, preparation method and applications thereof in preparation of genetic engineering drugs
  • Novel natriuretic peptide, preparation method and applications thereof in preparation of genetic engineering drugs

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preparation example Construction

[0031]The preparation method of the genetically engineered novel natriuretic peptide (BDNP) of the present invention comprises the following steps: A: obtain the BNP gene and the snake natriuretic peptide gene respectively; Gene splicing to synthesize a new natriuretic peptide gene; the new natriuretic peptide gene is composed of the brain natriuretic peptide gene at its N-terminus and the snake natriuretic peptide gene at its C-terminus; C: The expression plasmid including the new natriuretic peptide gene is constructed using a prokaryotic expression vector; D : Transform the expression plasmid formed in step C into an expression strain for induction culture to express a novel natriuretic peptide. After the expression is induced, the above method further includes step E: collecting the cells of the expressed strain by centrifugation, and purifying to obtain the genetically engineered novel natrium peptide. The method is simple and convenient to operate, and is convenient for ...

Embodiment 1

[0054] The new natriuretic peptide (BDNP) gene amplified by PCR splicing and sequencing was cloned into the prokaryotic expression vector pGEX-4T-1, and the prokaryotic expression plasmid pGEX-BDNP containing the new natriuretic peptide (BDNP) was constructed. BL21 and Rosetta were used as expression strains. Extract high-quality and high-concentration prokaryotic expression plasmid pGEX-BDNP, using cold CaCl 2 The above plasmids were transformed into BL21 and Rosetta strains respectively, and cultured overnight at 37°C; then, the expression strains were picked and inoculated into LB medium (adding ampicillin, the final concentration was 100 μg / ml), and cultured overnight at 37°C The bacterial liquid was collected, and the protein was extracted after ultrasonic crushing for Western Blot detection.

Embodiment 2

[0056] The new natriuretic peptide (BDNP) gene amplified by PCR splicing and sequencing was cloned into the prokaryotic expression vector pCW, and the prokaryotic expression plasmid pCW-BDNP containing the new natriuretic peptide (BDNP) was constructed. BL21 and Rosetta were used as expression strains. Extract high-quality and high-concentration prokaryotic expression plasmid pCW-BDNP, using cold CaCl 2 The above plasmids were transformed into BL21 and Rosetta strains respectively, and cultured overnight at 37°C; then, the expression strains were picked and inoculated into LB medium (adding ampicillin, the final concentration was 100 μg / ml), and cultured overnight at 37°C The bacterial liquid was collected, and the protein was extracted after ultrasonic crushing for Western Blot detection.

[0057] In the above step E, the purification of the novel sodium peptide of Example 1-2 includes collecting the expression strain by centrifugation, and then separating the fusion protein...

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Abstract

The invention discloses a novel natriuretic peptide, which is prepared through a connection of a brain natriuretic peptide and a dendroaspis natriuretic peptide, wherein the brain natriuretic peptide is on the N terminus of the novel natriuretic peptide, the snake natriuretic peptide is on the C terminus of the novel natriuretic peptide, and the brain natriuretic peptide comprises a disulfide bond cyclic structure which is composed of two cysteine residues. The preparation method comprises the following steps: obtaining a brain natriuretic peptide gene and a snake natriuretic peptide gene, connecting the two genes together to synthesize a novel natriuretic peptide gene through a PCR method; constructing an expression plasmid comprising the novel natriuretic peptide gene; and converting the expression plasmid to a proper expression bacterial strain to carry out an induction culture to induce the expression of the novel natriuretic peptide. The novel natriuretic peptide has the characteristics of the brain natriuretic peptide and dendroaspis natriuretic peptide at the same time, the specific effect is prominently strengthened, and the half-life period of drug is prominently prolonged. The preparation method has the advantages of simple technology and practicality; the gene engineering drugs having the novel natriuretic peptide can effectively act on effector organs such as heart, blood vessel, and kidney, effectively protect the functions of the organs mentioned above, prevent and treat diseases such as heart disease, kidney failure, etc.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals and relates to a novel sodium peptide prepared based on genetic engineering technology. More specifically, the present invention relates to a genetically engineered novel natriuretic peptide (BDNP) composed of brain natriuretic peptide (BNP) and snake natriuretic peptide (DNP), a preparation method and its use in the preparation for treating heart disease. Application of genetic engineering drugs in vascular diseases and kidney diseases, etc. Background technique [0002] A variety of natriuretic peptides have been discovered so far, such as atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide, urinary natriuretic peptide and snake natriuretic peptide, etc. Although they have some structural differences, they have the following common physiological effects: natriuretic peptide binds to the peroxisome proliferator-activated receptor on the blood vessel wall,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70A61K38/22A61P9/00A61P13/12
Inventor 李凌云周兆平王晓红章刚林枫
Owner SHENZHEN UNIV
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