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Method for expressing recombinant neurotrophic factor fusion protein, recombinant neurotrophic factor fusion protein and application of recombinant neurotrophic factor fusion protein

A neurotrophic factor and fusion protein technology, applied in the field of functional proteins, can solve the problems of complicated operation, low expression level, and low activity, and achieve the effects of reducing production costs, improving secretion efficiency, and good biological activity

Pending Publication Date: 2022-02-22
安徽中盛溯源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the use of recombinant expression to achieve in vitro production of neurotrophic factors has been reported. For example, the use of prokaryotic expression of neurotrophic factors in E. Need to induce expression, multi-step purification after inclusion body expression, cumbersome operation, low expression level and low activity
At present, there are also reports on the recombinant expression of neurotrophic factors using eukaryotic expression systems. For example, a Chinese patent with the publication number CN1364812A titled human glial cell-derived neurotrophic factors and their derivatives and their applications reported that the target gene was expressed using the expression vector pSVT7. Transfected into CHO cells, screened for GDNF-expressing cell lines, and then purified by heparin affinity chromatography, ion exchange chromatography, etc., not only the operation is complicated, but also the yield is low

Method used

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  • Method for expressing recombinant neurotrophic factor fusion protein, recombinant neurotrophic factor fusion protein and application of recombinant neurotrophic factor fusion protein
  • Method for expressing recombinant neurotrophic factor fusion protein, recombinant neurotrophic factor fusion protein and application of recombinant neurotrophic factor fusion protein
  • Method for expressing recombinant neurotrophic factor fusion protein, recombinant neurotrophic factor fusion protein and application of recombinant neurotrophic factor fusion protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Construction of high expression plasmids pPBml-PNC-TPA-Fc-GDNF and pPBml-PNC-TPA-BDNF-Fc

[0078] 1.1 Plasmid digestion

[0079] Take the existing pPBml-PNCE plasmid and the artificially synthesized TPA-Fc-GDNF or TPA-BDNF-Fc fusion protein sequence (synthesized by Nanjing GenScript) and digested at 37°C for 2h respectively. The digestion system is shown in Table 5 and Table 5. 6, the endonucleases include Xba I (manufacturer: NEB, product number: R0145L) and BamHI (manufacturer: NEB, product number: R3131L), and Cutsmart is a component in the endonuclease kit.

[0080] Table 5 pPBml-PNCE plasmid digestion system

[0081] component volume XbaI 3μL BamHI 3μL pPBml-PNCE 10μL Cutsmart 3μL ddH 2 O

11μL

[0082] Table 6 fusion protein sequence digestion system

[0083] component volume XbaI 5μL BamHI 5μL TPA-Fc-GDNF or TPA-BDNF-Fc 30μL Cutsmart 5μL ddH 2 O

5μL

...

Embodiment 2

[0091] Construction and screening of high-expressing cell lines

[0092] 1. Electroporation and screening of positive clones

[0093] 1.1 Plasmid electroporation

[0094] Select stable growth HEK293 cells (purchased from Zhuhai Kairui Biotechnology Co., Ltd.), the viability is greater than 95%, take 1 × 10 6 Cells were obtained by centrifugation to remove the supernatant. Resuspend the cells with 100 μL of electroporation buffer 10, add 1 μg pPBml-PNC-TPA-Fc-GDNF or pPBml-PNC-TPA-BDNF-Fc and pEhyPBase plasmids, mix well, and set a blank cell control group (no plasmid), transfer Transfer to the electroporation cup, electroporate with a Lonza electroporator (adjusted to HEK293 ATCC mode), resuspend the cells in DMEM+10% FBS medium after electroporation, spread them in a 6-well plate, and place at 37°C, 5% CO. 2 concentration, and cultivated in a humidified incubator.

[0095] 1.2 Screening of positive clones

[0096] After 24 hours of adherent growth, the adherent HEK293 ce...

Embodiment 3

[0098] Recombinant plasmid pPBml-PNC-TPA-GDNF-Fc (for the spectrum, see Figure 1-2 ) and pPBml-PNC-TPA-Fc-BDNF (see Figure 2-2 ) construction method

[0099] According to the method of Example 1, the pPBml-PNCE plasmid and the synthetic TPA-GDNF-Fc or TPA-Fc-BDNF fusion protein sequence were used to construct a recombinant vector. Positive clones were obtained according to the method of Example 2.

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Abstract

The invention provides a method for expressing recombinant neurotrophic factor fusion protein, the recombinant neurotrophic factor fusion protein and application of the recombinant neurotrophic factor fusion protein, and belongs to the technical field of functional protein. The dimer GDNF / BDNF recombinant protein with high biological activity is obtained by constructing a high-expression vector, using a cell expression system and screening a high-expression cell strain, and the defects that recombinant GDNF / BDNF on the market is low in expression quantity, high in price, low in biological activity and the like are overcome. The obtained GDNF / BDNF has very important significance for basic research and clinical-grade cell culture in the aspects of subsequent nerve growth and development and the like.

Description

technical field [0001] The invention belongs to the technical field of functional proteins, and in particular relates to a method for expressing a recombinant neurotrophic factor fusion protein, a recombinant neurotrophic factor fusion protein and applications thereof. Background technique [0002] In 1993, Lin, LF et al. used oligonucleotide probes to discover and screen human gene library from rat glioma cell line B49 cells, isolated and cloned a neurotrophic factor, named glial Derived neurotrophic factor (GDNF) (Lin LT et al., 1993). GDNF is a neurotrophic factor with important biological functions secreted by glial cells in brain tissue. It is a member of the TGF-beta superfamily and has the effect of promoting neuron growth and differentiation. Neurons show potential neurotrophic effects (Henderson et al., 1994), and also play an important role in participating in programmed neuronal cell death, promoting neuronal survival, and participating in repair of axonal damage...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/85C12N5/10C07K19/00C12N5/0793
CPCC07K14/475C12N15/85C12N5/0619C07K2319/30C07K2319/02C12N2501/998
Inventor 赵永强甘振磊俞君英张颖胡青松焦璐琰
Owner 安徽中盛溯源生物科技有限公司
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