Method for preparing maturation peptide of morphogenesis protein - 2 of human bones

A morphogenetic protein and mature peptide technology, which is applied in the preparation methods of peptides, biochemical equipment and methods, and botanical equipment and methods, etc., can solve the problems of long human bone morphogenetic protein-2 being unable to realize industrialized production, and achieve the cost The effect of low, high expression and simple production process

Active Publication Date: 2007-07-25
杭州北斗生物技术有限公司
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Problems solved by technology

[0017] In order to solve the technical defect that the long human bone morphogenetic protein-2 cannot be industrialized, the purpose of the present invention is to provide a hum

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  • Method for preparing maturation peptide of morphogenesis protein - 2 of human bones
  • Method for preparing maturation peptide of morphogenesis protein - 2 of human bones

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Embodiment Construction

[0047] 1. Acquisition of BMP-2 gene

[0048] Take 0.5 g of fetal liver tissue, grind it with a homogenizer, and extract total RNA with Trizol reagent. PCR primers were designed according to the BMP-2 gene sequence published by Genebank:

[0049] Primer 1: 5'TATA CATATG (NdeI restriction site) CAA GCC AAA CAC AAA CAGCGG.

[0050] Primer 2: 5'TGCA GAATTC (EcoR I restriction site) CGT CGACTC TAGAGTATA.

[0051] When designing the sequence, before the start codon ATG, an Nde I restriction site was added, and at the 3' end of the gene was a non-coding region sequence, so that the gene 3' end stop codon and the regulatory sequence stopped translation during expression.

[0052] Reverse transcription to obtain BMP-2 cDNA: In a 0.5ml micro-reaction tube, add 200pmol / L 3' end primer and 15μg total RNA to 24μl reaction solution, heat at 70°C for 5 minutes, add first-strand buffer, RNsin, AMV reverse Enzymes, dNTPs, and the mixed reaction were reacted at 42°C for 1 hour.

[0053] ...

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Abstract

This invention relates to a method for preparing mature human bone morphogenetic protein-2. The method comprise: (1) obtaining mature human bone morphogenetic protein-2 gene; (2) constructing expression vector, and transferring Escherichia coli; (3) screening to obtain positive clones, culturing and inducing to express; (4) collecting the inclusion body, and renaturing; (5) separating and purifying the expression product. The expression vector is pET series expression vector. The method has such advantages as high expression level, simple process, and low cost. The product has bioactivity, and can be used to accelerate bone tissue regeneration.

Description

technical field [0001] The invention relates to the technical field of biosynthesis, in particular to a preparation method of human bone morphogenetic protein-2 mature peptide. Background technique [0002] In 1965, American doctor Urist discovered that decalcified bone mesenchyme can induce ectopic osteogenesis. He believed that there is a substance in decalcified bone mesenchyme that can induce new bone formation, and named it bone morphogenesis. Protein (Bone Morphogenetic Protein BMP). In 1988, Wozney et al. used recombinant DNA technology to clone the cDNAs of four human BMPs for the first time in the world, and their discoveries received great attention from scientists all over the world. In 1990, Celeste et al. reported the cloning research of the other three BMPs, and suggested that the cloned BMPs be named BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, and BMP-6, respectively. , BMP-7. Except for BMP-1, they are all members of TGF-β superfamily, and they are very similar in ...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/12C12N15/70C12P21/02C07K1/14
Inventor 方永强刘国安
Owner 杭州北斗生物技术有限公司
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