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CHO cell strain capable of stably and efficiently expressing recombinant human BMP7 (bone morphogenetic protein-7) and medical application

A high-efficiency expression and cell line technology, applied in the field of bioengineering, can solve the problems of increasing the complexity and cost of downstream purification processes, high cost of cell culture, and difficult conditions, etc., to achieve gene compatibility and translation efficiency, and improve transcription and translation Efficiency and the effect of maintaining stability

Active Publication Date: 2017-09-15
长春生物制品研究所有限责任公司
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Problems solved by technology

However, the cost of CHO cell culture is high, the conditions are difficult to master, easy to contaminate, and the expression level is low
Serum components in the culture medium are the main factors causing high production costs. Serum is expensive, inconsistent in quality between batches, and contains animal-derived components, which greatly increase the complexity and cost of downstream purification processes

Method used

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  • CHO cell strain capable of stably and efficiently expressing recombinant human BMP7 (bone morphogenetic protein-7) and medical application
  • CHO cell strain capable of stably and efficiently expressing recombinant human BMP7 (bone morphogenetic protein-7) and medical application
  • CHO cell strain capable of stably and efficiently expressing recombinant human BMP7 (bone morphogenetic protein-7) and medical application

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Experimental program
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Embodiment 1

[0029] 1. Acquisition of mouse bmp7 gene

[0030] Take the bone tissue of a 1-day-old mouse and put it in liquid nitrogen for 30 minutes, take 200 mg and put it in a grinder, add 2 ml TRizol, grind it in an ice bath, transfer the suspension into a 1.5 ml centrifuge tube, and let it stand at room temperature for 5 minutes. Take 1ml of clarified tissue fluid, add 200μl chloroform, shake vigorously and let stand on ice for 5 minutes, centrifuge at 12000rpm at 4°C for 10 minutes, absorb 0.6ml of the upper aqueous phase, add 0.6ml of isopropanol, mix well and let stand on ice for 10 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes, discard the supernatant, wash the RNA pellet with 75% ethanol, centrifuge at 12,000 rpm at 4°C for 5 minutes, place the RNA pellet at room temperature to dry, and add 100 μl of RNase-free water to dissolve it. Take 1 μl of RNA and apply TaKaRa RNA PCR kit to reverse transcribe into cDNA. The reverse transcription conditions were 60°C for 30 minut...

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Abstract

The invention provides a CHO cell strain capable of stably and efficiently expressing recombinant human BMP7 (bone morphogenetic protein-7) and a medical application. A bmp7 gene sequence is constructed by modifying a bmp7 gene of a mouse, a CHO cell serving as a host is also derived from the mouse, the genetic compatibility and the translation efficiency are greatly improved, the defect that gene sequence templates of BMP7 protein expressed by a CHO system are all derived from human bmp7 genes in the prior art is overcome, and the situation that human-sourced gene sequences are difficult to transcribe and translate efficiently by CHO cells due to codon preference is avoided; meanwhile, the adopted CHO-S cells are suspension cultured cells which have been adapted to a serum-free culture medium, the cell strain does not need to be subjected to suspension acclimation, and the engineering CHO cell strain can keep the stability conveniently. The adopted technical means has a remarkable efficiency improvement function in key steps of BMP expression as follows: transcription, translation, folding, conditioned cleavage, secretion, pairing of disulfide bonds and glycoform modification of glycosylation sites.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and provides a CHO cell strain integrated with a modified mouse bmp7 gene, which is a CHO cell strain stably and efficiently expressing recombinant human BMP7, and also discloses its medical application. Background technique [0002] Bone Morphogenetic Proteins (BMPs) are a class of low species-specific protein growth factors with multiple subtypes. BMPs can induce the irreversible differentiation of mesenchymal cells into chondrocytes and osteocytes. Bone morphogenetic protein-7, also known as osteogenic protein-1 (OP-1), participates in the proliferation and differentiation of cells during the embryonic period, and regulates the formation and development of kidneys, intestines, bones and other organs. Adult BMP7 is mainly expressed in bone and kidney tissues, especially during bone development and fracture healing. [0003] BMP7 is a member of the TGF-β superfamily. Its precursor prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/12C12N15/85C12P21/00C12R1/91
CPCC07K14/51C12N15/85
Inventor 刘畅郭凤祥于爱东宋昊韩顺子唐剑光刘涵周利微关诗宇于虹常东英吴晓娟迟春萍曹玉锋时成波李利刘照惠
Owner 长春生物制品研究所有限责任公司
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