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Mature peptide of recombination human DPT (dermatopontin) and production method thereof

A production method and technology for mature peptides, which are applied in the field of highly efficient recombinant human skin pontin mature peptides and their production fields, can solve the problem that the mechanism of action needs to be further studied, and achieve the effects of being easy to purify and detect, reducing metabolic load, and promoting folding

Inactive Publication Date: 2017-08-18
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In conclusion, DPT is an important secreted protein that widely exists in the cell matrix. It has functions such as cell adhesion and promotion of collagen fiber formation through the actions of decorin, TGFβ, and type I collagen. However, the mechanism of action of DPT in the physiological and pathological processes related to extracellular matrix remains to be further studied
At present, there is no relevant report on the large-scale expression and purification of DPT in vitro

Method used

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  • Mature peptide of recombination human DPT (dermatopontin) and production method thereof
  • Mature peptide of recombination human DPT (dermatopontin) and production method thereof
  • Mature peptide of recombination human DPT (dermatopontin) and production method thereof

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Embodiment 1

[0045] In this embodiment, a high-efficiency rhDPT expression and purification method mainly includes the following steps:

[0046] 1. Clone the whole rhDPT gene:

[0047] Design a pair of DPT characteristic amplification primers, and use artificially synthesized DPT cDNA as a template to specifically amplify the complete human skin bridge protein mature peptide gene fragment (rhDPT gene), and the 5' end of the rhDPT gene is introduced into the EcoR1 restriction site , the 3' end introduces the Not1 restriction site (the base sequence of the rhDPT gene that introduces the restriction site is shown in SEQ ID NO.1) so that the subsequent insertion of the rhDPT gene fragment into the expression vector; PCR conditions are: 95 ° C pre- Denaturation, 5min, one thermal cycle; thermal denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, 30 thermal cycles; renaturation at 72°C for 10min. The amplified rhDPT gene fragment was recovered and amplified by ag...

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Abstract

The invention discloses mature peptide of recombination human DPT (dermatopontin) and a production method thereof. The production method comprises the following steps of obtaining a segment gene of the mature peptide of the human DPT; establishing an eukaryotic expression carrier, and converting into pichia pastoris X-33; performing resistance screening to obtain a yeast transformant with high-level excretion expression; inducing to express supernatant, and purifying by a nickel column, so as to obtain the rhDPT. The recombination protein rhDPT prepared by the method has the advantages that the HIS and C-MYC labels are carried, and the protein purification and detection are easy; in the in-vitro experiment, the rhDPT can effectively inhibit the growth of fat cells, and the biological activity is good.

Description

technical field [0001] The invention relates to the technical field of using recombinant DNA technology to produce genetically engineered protein drugs, in particular to a high-efficiency recombinant human skin bridge protein mature peptide (rhDPT) and a production method thereof. Background technique [0002] The dermatopontin (DPT) gene is located on human chromosome 1q12-1q23. It is a secreted protein composed of 183 amino acid residues, with a relative molecular mass of 22kDa. DPT is rich in tyrosine residues and 1 / 2 is sulfated. The sulfation of tyrosine may be related to the effect of DPT on the formation of collagen fibers and its binding to cell surface receptors. DPT is widely distributed in the tissues of mammals. DPT exists in skin, bone, heart, lung, kidney, cartilage, and bone of pigs and rodents. DPT in humans is mainly distributed in muscle, heart, pancreas, lung, etc. in tissue fibroblasts. The latest research found that DPT is also distributed in the extra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C07K14/47C07K1/22
CPCC12N15/815C07K14/47
Inventor 吴东海孙伟聂涛李快韩卫平
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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