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Method for expressing recombinant human bone morphogenetic protein in insect cell

A morphogenetic protein, insect cell technology, applied in animal/human proteins, biochemical equipment and methods, botanical equipment and methods, etc., can solve the problems of low protein expression, high treatment cost, and high preparation cost

Inactive Publication Date: 2012-11-14
YANGZHOU TAIDA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the low expression of recombinant human bone morphogenetic protein prepared from animal cells and the high cost of preparation, the treatment cost is as high as 6,000-10,000 US dollars, which greatly limits the clinical application of BMP

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  • Method for expressing recombinant human bone morphogenetic protein in insect cell
  • Method for expressing recombinant human bone morphogenetic protein in insect cell
  • Method for expressing recombinant human bone morphogenetic protein in insect cell

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Embodiment Construction

[0016] Below in conjunction with embodiment, the present invention is further described. The following examples are illustrative rather than limiting, and the protection scope of the present invention cannot be limited by the following examples.

[0017] The steps of the method for highly expressing mature peptides of recombinant human bone morphogenetic proteins BMP2 and BMP7 in insect cells are as follows:

[0018] (1) Preparation and identification of recombinant baculovirus

[0019] Respectively in human BMP2, BMP7 mature peptide ( figure 1 ) with a 6xHis tag added to the N-terminus, and its gene fragments were cloned into an improved insect-baculovirus expression vector with BamHI and EcoRI sites, and then co-transfected into sf9 insect cells. Clear culture fluid, lyse baculovirus with proteinase K, carry out PCR with viral genome DNA as a template, and amplify the DNA fragments encoding BMP2 and BMP7 mature peptide respectively ( figure 2 ), indicating that a recombi...

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Abstract

The invention relates to a method for expressing a recombinant human bone morphogenetic protein in insect cells. The method comprises the following steps of cloning a mature peptide gene segment of the human bone morphogenetic protein (BMP) to an an improved insect cell-baculovirus protein expression vector; co-transfecting sf9 insect cells; preparing recombinant baculovirus having mature peptide gene segment of the BMP; then transfecting High5 insect cells; highly expressing the BMP mature peptides; and purifying the expressed BMP mature peptides via an Ni affinity column. Detections on in-vitro alkaline phosphatase activity and induced-bone experiments of muscular pouch embedded in mice demonstrate that both expressed BMP2 and BMP7 have activities for inducing ectopic bone formation. By using an improved insect cell-baculovirus protein expression system,, 40 mg purified BMP2 or BMP7 mature peptide can be obtained per litre cultured insect cells; and cost is reduced at least 10 times than that in CHO cells.

Description

technical field [0001] The invention relates to a new technology method for highly expressing biologically active recombinant human bone morphogenetic proteins BMP2 and BMP7 in insect cells by using an insect cell-baculovirus protein expression system. Background technique [0002] Bone morphogenetic protein (BMP) is a protein that was first discovered in 1965 and can induce normal and ectopic osteogenesis. Ability to induce new bone formation. More than 20 BMPs have been identified and cloned, which belong to the transforming growth factor β (transforming growth factor, TGF-β) supergene family members. BMP2 is one of the most widely studied BMPs with the strongest osteogenic activity. BMP7 is mainly expressed in bone and kidney, has strong osteoinductive activity, can stimulate the proliferation of undifferentiated mesenchymal cells and their differentiation into osteoblasts or chondrocytes, and promote the proliferation of osteoblasts and alkaline phosphatase secretion....

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/866C12N7/01C07K14/495C07K1/22
Inventor 刁爱坡王飞许丽红罗深恒
Owner YANGZHOU TAIDA BIOTECH
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