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Long chain recombinant human bone morphogenesis protein-2 and its preparation method and uses

A morphogenetic protein and long-chain technology, applied in bone morphogenetic factors, biochemical equipment and methods, animal/human proteins, etc., can solve the problems of low protein activity, instability, and difficulty in industrialization

Active Publication Date: 2010-05-12
SHANGHAI REBONE BIOMATERIALS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The technical problem to be solved in the present invention is to disclose a long-chain recombinant human bone morphogenetic protein-2 and its preparation method and application, so as to overcome the difficulty in renaturation, separation, low protein activity and instability in the body in the prior art. Causes problems that are difficult to industrialize

Method used

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  • Long chain recombinant human bone morphogenesis protein-2 and its preparation method and uses
  • Long chain recombinant human bone morphogenesis protein-2 and its preparation method and uses
  • Long chain recombinant human bone morphogenesis protein-2 and its preparation method and uses

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Experimental program
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Effect test

Embodiment 1

[0191] Embodiment 1 long-chain type rhBMP-2 and its preparation method

[0192] ①Cultivate the human osteosarcoma cell line U20S (purchased from ATCC, USA), collect the cells, lyse the cells to extract mRNA, use Oligo(dT) as a primer to reverse transcribe into cDNA, artificially synthesize the primer, and add the first codon at the 5' end of the forward primer Add a nucleotide sequence encoding the designed amino acid residue sequence (Met Lys Arg His Asp Gly Lys Gly His Pro LeuHis Lys Arg Glu Lys Arg) before the substring, and then carry out PCR amplification using cDNA as a template to obtain a It is a 396bp DNA fragment. The above fragment is recovered and inserted into the expression vector pBV220 to construct a long-chain recombinant human BMP-2 gene expression vector. After sequencing verification, its gene coding sequence is shown in SEQ ID: NO:4. The amino acid residue sequence of the long-chain recombinant human BMP-2 is shown in SEQ ID: NO:1.

[0193] ② Molecular cl...

Embodiment 2

[0198]Embodiment 2 long-chain type rhBMP-2 and its preparation method

[0199] ①Cultivate the human osteosarcoma cell line U20S (purchased from ATCC, USA), collect the cells, lyse the cells to extract mRNA, use Oligo(dT) as a primer to reverse transcribe into cDNA, artificially synthesize the primer, and add the first codon at the 5' end of the forward primer Add a nucleotide sequence encoding the designed amino acid residue sequence (Met Gly His Pro Leu His Lys Arg Glu LysArg) before the substring, and then carry out PCR amplification using cDNA as a template to obtain a DNA fragment with a size of 378bp. The above fragment was recovered and inserted into the expression vector pBV220 to construct a long-chain recombinant human BMP-2 gene expression vector. After sequencing verification, the gene coding sequence is shown in SEQ ID: NO:5. The amino acid residue sequence of the long-chain recombinant human BMP-2 is shown in SEQ ID: NO:2.

[0200] ② Molecular cloning techniques ...

Embodiment 3

[0205] Embodiment 3 long-chain type rhBMP-2 and its preparation method

[0206] ①Cultivate the human osteosarcoma cell line U20S (purchased from ATCC, USA), collect the cells, lyse the cells to extract mRNA, use Oligo(dT) as a primer to reverse transcribe into cDNA, artificially synthesize the primer, and add the first codon at the 5' end of the forward primer Add a nucleotide sequence encoding the designed amino acid residue sequence (Met Arg Glu Lys Arg) before the substring, then carry out PCR amplification using cDNA as a template to obtain a DNA fragment with a size of 360bp, reclaim the above fragment, and It is inserted into the expression vector pBV220 to construct a long-chain recombinant human BMP-2 gene expression vector. After sequencing verification, the gene coding sequence is shown in SEQ ID: NO:6. The amino acid residue sequence of the long-chain recombinant human BMP-2 is shown in SEQ ID: NO:3.

[0207] ② Using the molecular cloning technique, the competent c...

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Abstract

The invention discloses a long-chain recombination human bone pattern generating protein-2 and preparing method and application, which is characterized by the following: utilizing gene project technique to grow the protein in the expressing system of escherichia coli and bacillus subtilis; adopting human bone sarcoma cell mRNA to do reverse transcription to obtain the cDNA as form; augmenting nucleotide sequence of entire 114 amino acids naturally from carboxyl end; adding a segment of nucleotide in front of the first codon of primer 5' end; increasing a segment of polypeptide at N end corresponding to amino acid sequence; obtaining long-chain rhBMP-2 gene with molecular weight at 30KD and purity over 95%.

Description

technical field [0001] The invention relates to a recombinant human bone morphogenetic protein-2 and its genetic engineering preparation method. Background technique [0002] Delayed fracture union, bone nonunion, and repair of bone defect have always been major unresolved problems in the field of orthopedics. For a long time, the repair of bone defects has mainly adopted three treatment methods: autologous bone graft, allogeneic bone graft, and replacement and repair with biomaterials and products. Autologous bone is limited by the number of sources, and there are at least 10% surgical complications in bone harvesting operations, and a long crawling replacement process is required after implantation; allogeneic bone has different degrees of immune rejection and potential risk of disease transmission. Therefore, biomaterials and products with special functions have attracted much attention clinically. Although many biomaterials have been used clinically, the lack of activi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/51C07K19/00C12N15/12C12N15/62C12N15/09C12N1/21A61L27/54A61P19/00
Inventor 刘昌胜林俊陈芳萍曹雪华
Owner SHANGHAI REBONE BIOMATERIALS
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