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Preparation method of human BMP2 (bone morphogenetic protein 2) and analogue thereof

A BMP2-T, DS-BMP2-T technology, applied in the field of genetic engineering, can solve the problems of complex process, low expression, low renaturation efficiency, etc., and achieve the effect of good process controllability and simple operation

Pending Publication Date: 2022-03-04
FIRST HOSPITAL AFFILIATED TO GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the CHO expressed protein can form a complete protein structure, the expression level is low and the production cost is high
At present, there are also reports based on the expression and renaturation of BMP2 in Escherichia coli, but the renaturation efficiency is generally low and the process is complicated.

Method used

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  • Preparation method of human BMP2 (bone morphogenetic protein 2) and analogue thereof
  • Preparation method of human BMP2 (bone morphogenetic protein 2) and analogue thereof
  • Preparation method of human BMP2 (bone morphogenetic protein 2) and analogue thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] The preparation of embodiment 1 BMP2-T

[0039] (1) BMP2-T coding gene synthesis, vector construction and expression strain construction

[0040] 1.1 The 290-396th position of the gene encoding the BMP2 protein (283-396th position of unipro No.P12643), and introduce the R291K mutation modification, and replace the codon with the E. coli preference ( High-frequency use) codons to be suitable for expression in Escherichia coli, and add the initiation codon ATG before the optimized sequence, and finally obtain the optimized BMP2-T coding gene sequence as shown in the sequence table SEQ ID NO: 1 shown.

[0041] 1.2 Insert the double-stranded DNA molecule artificially synthesized by the coding strand nucleotides shown in SEQ ID NO: 1 in the sequence table through the principle of complementary base pairing into the NdeI / BamHI restriction site of the vector pET11a(+) to obtain a recombinant plasmid ( figure 1 ). In the recombinant plasmid, the inserted double-stranded DNA...

Embodiment 2

[0068] The preparation of embodiment 2 BMP2

[0069] 2.1 BMP2 encoding gene synthesis, vector construction and expression strain construction

[0070]The 283-396th position of the gene encoding the BMP2 protein (283-396th position of unipro No.P12643), without changing the amino acid sequence, replaced its codon with the preferred codon of Escherichia coli to be suitable for the large intestine Bacteria, and the start codon ATG was added before the optimized sequence to obtain the optimized BMP2 coding gene sequence as shown in the sequence table SEQ ID NO:3.

[0071] Insert the double-stranded DNA molecule artificially synthesized by the coding chain nucleotides shown in SEQ ID NO: 3 in the sequence table through the principle of complementary base pairing into the NcoI / XhoI restriction site of the vector pET-28a(+) to obtain the recombinant Plasmid ( Figure 8 ). In the recombinant plasmid, the inserted double-stranded DNA molecule is fused with part of the DNA of the pla...

Embodiment 3

[0082] Example 3 Preparation of DS-BMP2-T

[0083] 3.1 DS-BMP2-T coding gene synthesis, vector construction and expression strain construction

[0084] The 290-396th position of the gene encoding BMP2 protein (283-396th position of unipro No. P12643) is introduced into the R291K mutation modification. In order to improve the binding force of the BMP2 mutant to the collagen in the bone matrix, on the basis of the core protein sequence, a bone-binding polypeptide (sequence is DSSSSSSSSSSSSSSS) was introduced at the N-terminus of the protein, and the bone-binding polypeptide and the BMP2-T sequence The linking region of the protein was introduced into the linking sequence (sequence GSGS), in order to mention the expression of the protein, the high expression of the protein was finally obtained by fusing and expressing the form of TRX tag (thioredoxin protein) at the N-terminus. The final expressed protein sequence is shown in SEQ ID NO:6. Under the premise of not changing the a...

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Abstract

The invention discloses a preparation method of human-derived BMP2 (bone morphogenetic protein 2) and analogues thereof. The method comprises the following steps: firstly, introducing a coding gene of human-derived BMP2 or an analogue thereof into a receptor escherichia coli cell; after carrying out induced expression on the recombinant escherichia coli cells, collecting and crushing to obtain an inclusion body; denaturating the inclusion body to obtain denatured protein; and carrying out renaturation and purification on the denatured protein to obtain the human-derived BMP2 and the analogue thereof. According to the invention, escherichia coli is used as an expression system, BMP2 analogues composed of different sequences are designed and constructed based on the theoretical sequence of BMP2, and finally, a production process of mutant protein suitable for industrial production is obtained. The production process has the characteristics of simple operation, good process controllability and large-scale production.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to prokaryotic recombinant expression, preparation, in vitro activity detection and clinical application of human BMP2 cytokine. Background technique [0002] Fracture repair is a slow and painful process, and clinical research continues to promote the development of drugs for fracture repair. Bone morphogenetic protein (Bone morphogenetic protein) is a kind of transformation production factor (TGF-β) secreted by bone matrix. It was first proposed by Uris et al. in 1965, and was first isolated from the demineralized bone matrix of bovine bone in 1982. So far, eight family members (BMP2, BMP4, BMP5, BMP6, BMP7, BMP9, BMP12, BMP14) have been found. Among them, BMP2 has the obvious effect of inducing osteocyte differentiation, thereby promoting the formation of heterotopic solids and promoting fracture repair. [0003] BMP2 protein has the typical characteristics of TGF-β family prot...

Claims

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Application Information

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IPC IPC(8): C07K14/51C07K19/00C07K1/22C07K1/20C07K1/18C07K1/16C12N15/12C12N15/70
CPCC07K14/51C12N15/70C12N2800/101C12N2800/22C07K2319/00
Inventor 赵彦涛胡先同韩丽伟李利李岩峰郭继东王华东洪磊
Owner FIRST HOSPITAL AFFILIATED TO GENERAL HOSPITAL OF PLA
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