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Recombinant expression vector for human beta-NGF and recombinant cell strain containing same

A technology for expression vectors and eukaryotic cells, applied to cells modified by introducing foreign genetic material, recombinant DNA technology, and the use of vectors to introduce foreign genetic material. Antibody response and other issues, to achieve the effect of uniform drug resistance, high cell drug resistance, and uniform expression

Active Publication Date: 2013-05-01
深圳市中科深研生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are two main methods for producing β-NGF: one is β-NGF extracted from the submandibular gland of mice with a molecular weight of about 13-14KD and a sedimentation coefficient of 2.5S. The β-NGF obtained in this way has the following disadvantages: (1) There is a 10% difference in protein sequence between mouse NGF and human NGF, which makes it immunogenic and may induce antibody responses, thereby reducing the efficacy of the drug; (2) There is a potential safety hazard of cross-infection of mouse-derived viruses; (3) ) production depends on live animals
The other is prepared by recombinant expression of prokaryotic expression system (E.coli). The defect of this method is that the protein cannot undergo post-translational modification, and the activity of the obtained β-NGF is significantly lower than that of the natural β-NGF

Method used

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  • Recombinant expression vector for human beta-NGF and recombinant cell strain containing same
  • Recombinant expression vector for human beta-NGF and recombinant cell strain containing same
  • Recombinant expression vector for human beta-NGF and recombinant cell strain containing same

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1 Construction of β-NGF recombinant expression plasmid of the present invention

[0033] 1. Cloning of human β-NGF gene and construction of pCMV-β-NGF recombinant vector

[0034] (1) Total RNA was extracted from human placental cells using TriZol.

[0035] (2) Using the total RNA obtained in step (1) as a template, use Superscript III reverse transcriptase to synthesize a cDNA library by reverse transcription.

[0036] (3) Using the cDNA library obtained in step (2) as a template, use primers that specifically amplify the full length of the β-NGF gene, and perform PCR amplification under the catalysis of pfx DNA polymerase; the PCR reaction conditions are: 94°C pre-denaturation 3min; 30 cycles of denaturation at 94°C for 30s, annealing at 55°C for 30s, and extension at 68°C for 1min.

[0037]The upstream and downstream primers for specifically amplifying the full length of the β-NGF gene are respectively shown in SEQ ID No.4 and SEQ ID No.5. The upstream and d...

Embodiment 2

[0049] Example 2 Construction of β-NGF recombinant cell line of the present invention

[0050] 1. Transfection and stable screening of NIH293 cells

[0051] (1) Plasmid purification: The pCMV-β-NGF-IRES-dhfr plasmid prepared in Example 1 was purified by cesium chloride density gradient centrifugation and kept for future use.

[0052] (2) Transfection and screening of recombinant cell lines: Use DEME complete medium (containing 10% calf serum) at 37°C, 5% CO 2 NIH293 cells were cultured to a 60% monolayer under the environment of NIH293 cells, and the above-mentioned purified pCMV-β-NGF-IRES-dhfr plasmid was transfected into NIH293 cells by calcium phosphate co-precipitation method; after 24 hours, the medium was changed, and 50nM MTX was added to filter. After the cells adapt to the selection pressure, the concentration of MTX is gradually increased, which is 100nM, 200nM, 400nM and 800nM. After the cells are adapted to 800nM MTX, further increase the MTX concentration to 1...

Embodiment 3

[0056] Example 3 Identification of β-NGF secreted by the β-NGF recombinant cell line of the present invention

[0057] A β-NGF recombinant cell line obtained in Example 2 was cultured without serum, the medium was collected and the protein was concentrated to obtain β-NGF, and the β-NGF was identified by mass spectrometry.

[0058] Mass spectrometric identification method:

[0059] The obtained β-NGF was separated by capillary liquid chromatography-electrospray ionization-quadrupole-time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS) for peptide map determination: β-NGF was hydrolyzed with trypsin and dissolved in 0.1% (v / v) formic acid solution was analyzed by reversed-phase nanoliter liquid chromatography-electrospray tandem mass spectrometry. Using BEH130 capillary liquid chromatography, load 10 μl of the sample to the enrichment column (180 μm×20mm Symmetry C18trap column), desalt with buffer A (that is, 0.1% formic acid aqueous solution) at a flow rate of 10 μl / min for ...

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Abstract

The invention discloses a recombinant expression vector for stably and efficiently expressing a human beta-NGF (Nerve Growth Factor), a recombinant cell strain containing the recombinant expression vector and a method for producing a recombinant human beta-NGF. The recombinant expression vector comprises a promoter, a beta globin gene intron and a human beta-NGF gene, an internal ribosome entry site sequence and a selective marker gene and has higher integration and expression efficiency in a host cell. A recombinant eukaryotic cell containing the recombinant expression vector expresses the recombinant human beta-NGF equivalent with a natural beta-NGF in activity and still can stably express after multiple times of passage; and the expressed beta-NGF is secreted into a culture medium and is easy to separate and purify, the production downstream difficulty and the production downstream cost are greatly reduced, and the clinical application prospect and the commercial value are good.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a secreted expression vector of recombinant human β-NGF and a recombinant cell strain containing the vector. Background technique [0002] Nerve Growth Factor (NGF) is the earliest neurotrophic factor discovered and currently the most thorough study of a nerve cell growth regulator. It has dual biological functions of neuron nutrition and neurite growth. , differentiation, growth, regeneration, and expression of functional properties all have important regulatory roles. NGF is mainly distributed in the brain, ganglion, iris, heart, spleen, placenta and other tissues in the human body, as well as fibroblasts, smooth muscle, skeletal muscle, glial cells, Schwann cells, etc. NGF binds to the receptor and is internalized through the receptor-mediated endocytosis mechanism to form vesicles surrounded by the axon membrane, containing NGF, and maintaining its biological activity, which are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C07K14/48
Inventor 薛博夫马墨丽贝卡·梅尔本朱林
Owner 深圳市中科深研生物科技有限公司
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