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NGF-Fc fusion protein and preparation method thereof

A fusion protein and immunoglobulin technology, which is applied in the field of highly efficient expression of NGF-Fc fusion protein, can solve the problems of prolonging plasma half-life and failing to enhance protein stability, and achieve the effects of druggability, prolonging half-life and improving expression level

Inactive Publication Date: 2016-01-27
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of Fc to prepare fusion proteins has been reported, but there are defects in the fusion proteins that cannot enhance protein stability and prolong plasma half-life

Method used

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  • NGF-Fc fusion protein and preparation method thereof
  • NGF-Fc fusion protein and preparation method thereof
  • NGF-Fc fusion protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of NGF-Fc fusion gene

[0039] Preparation of DNA sequence encoding NGF peptide: artificially synthesized DNA sequence encoding NGF peptide, and introduced NheI and BamHI restriction sites at both ends of the DNA sequence. Use NheI and BamHI double enzyme digestion system to carry out enzyme digestion and purification to obtain the NGF fragment with NheI-BamHI sticky end, and carry out double enzyme digestion and purification with NheI and BamHI on the transition plasmid pUC-Fc containing the DNA sequence encoding the Fc peptide. Obtain the DNA carrier fragment with NheI-BamHI cohesive end, connect the NGF fragment and the vector fragment, transform Escherichia coli, obtain the positive clone pUc-NGF-Fc, and confirm it by enzyme digestion and sequencing.

Embodiment 2

[0040] Example 2 Preparation of NGF-Fc fusion gene expression vector

[0041] pUc-NGF-Fc was double digested with NdeI and NotI, the NGF-Fc fragment was recovered from gel sugar, the expression vector was double digested with NdeI and NotI, the large fragment (vector fragment) was recovered from gel sugar, and ligated with T4DNA ligase Carrier DNA fragments and NdeI and NotI fragments, and the ligated products were transformed into Escherichia coli, and positive clones were obtained by selection and identification, and the enzyme digestion identification was correct.

Embodiment 3

[0042] Embodiment 3 NGF-Fc fusion protein expression and purification

[0043] Transfection of NGF-Fc Fusion Expression Vector into CHO Cells Lipofectamine-mediated CHO Cell Transfection: Culture CHO cells to 40%-60% confluence. Prepare transfection solution: Solution A: Dilute 10 μg DNA to 100 μl with serum-free DMEM medium; Solution B: Dilute 15 μl Lipofectamine with serum-free DMEM medium to 100 μl; Gently mix A and B two solutions, leave at room temperature for 15 minutes, and slowly add In the cell culture dish, shake well, place in a CO2 incubator at 37°C for 24 hours, aspirate the supernatant, and replace with complete medium to continue culturing;

[0044] Screening of positive cells: Aspirate the culture medium, replace it with the screening medium ((10% calf serum DMEM complete medium containing 800 μg PmlG418 containing 800 μg PmlG418) for screening high-expression monoclonal cell lines, and replace it with the screening medium after most of the cells die Maintenan...

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Abstract

The invention belongs to the technical field of biological engineering, and relates to an NGF-Fc fusion protein and a preparation method thereof. The method comprises the following steps: constructing a human immune globulin IgG Fc and nerve growth factor (NGF) fusion gene expression vector through a genetic engineering means, transferring to a mammal cell to make the transferred mammal cell highly express and produce NGF-Fc fusion proteins, purifying, identifying and carrying out biological activity detection. The expression vector transferred mammal cell constructed in the invention can express bioactive NGF-Fc fusion proteins, so the expression level can be 150mg / L or above, and the obtained protein has good stability and long half life, and can be used to prepare drugs for treating Alzheimer's disease, diabetic peripheral neuropathy, Parkinson's disease, facial neuritis, craniocerebral trauma, trauma of spinal cord, acute cerebrovascular disease, encephalatrophy and other neurological diseases, and peripheral nerve injury acute cerebral vascular central nerve injuries induced by chemical drugs.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a NGF-Fc fusion protein and a preparation method thereof, in particular to a method for obtaining high-efficiency expression of the NGF-Fc fusion protein by constructing a recombinant engineering cell line. Background technique [0002] According to reports, nerve growth factor (NGF) is the earliest discovered neurotrophic factor, the most thorough research at present, a nerve cell growth regulator with dual biological functions of neuron nutrition and neurite growth. It plays an important regulatory role in the development, differentiation, growth, regeneration and expression of functional properties of peripheral neurons. Studies have shown that NGF contains three subunits of α, β, and γ, and the active region is the β subunit, which is a dimer composed of two single chains of 118 amino acids combined through non-covalent bonds, which has the same structure as human NGF. ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K38/18A61K47/48A61P25/28A61P25/16A61P25/02A61P25/00A61P9/10
Inventor 冯美卿叶丽鞠佃文许必雄郭颀然
Owner FUDAN UNIV
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