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Glial cell line-Derived Neurotrophic Factor (GDNF) fusing penetrating peptides

A trophic factor, nerve cell technology, applied in hybrid peptides, nervous system diseases, recombinant DNA technology, etc., can solve HIV-TAT safety concerns, application limitations and other issues

Active Publication Date: 2010-07-14
广州市凯诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] HIV-TAT-mediated protein transduction is subject to pre-denaturation to obtain high transduction rate and depends on intracellular molecular chaperone HSP90 disassembly to exert its biological effects, and more importantly, the safety of HIV-TAT concerns, so the application of this technology is limited

Method used

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  • Glial cell line-Derived Neurotrophic Factor (GDNF) fusing penetrating peptides
  • Glial cell line-Derived Neurotrophic Factor (GDNF) fusing penetrating peptides
  • Glial cell line-Derived Neurotrophic Factor (GDNF) fusing penetrating peptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: human GDNF gene cloning, gene fusion and its expression plasmid construction:

[0037] 1). Human GDNF gene cloning and fusion gene fusion:

[0038] Using the prepared human brain tissue information ribonucleotide (mRNA) as a template, use Poly(T) primers under the action of reverse transcriptase to synthesize complementary deoxyribonucleotide (cDNA) of human brain tissue; Brain tissue cDNA was used as a template, and the P-1-1 and P-1-2 forward primers containing the nucleotides encoding the penetrating peptide PEP-1 and the N-terminal sequence of the human GDNF gene and the human GDNF gene C - The reverse primer of the terminal nucleotide sequence is used for secondary PCR amplification, the human source GDNF gene is cloned, and its N-terminal modification is completed.

[0039] a: PEP-1 primers were designed as follows:

[0040] P-1-1:

[0041] 5'-tcgagctcaggaggaaacgccatggagaaagaaacctggtgggaaacctggtggaccg-3'

[0042] P-1-2:

[0043] 5'-cct...

Embodiment 2

[0049] Example 2: Expression of the expression plasmid pET-45b(+)-PEP-1 / GDNF in prokaryotic bacteria:

[0050] 1). Transformation of competent expression bacteria: Take 1-2 micrograms of pET-45b(+)-PEP-1 / GDNF prokaryotic expression plasmid DNA and add it to the competent expression bacteria BL21(DE3)PlysS thawed on ice, mix and place After incubating on ice for 30 minutes; then, in a water bath at 42°C for 42 seconds, and place on ice for 1 minute; add 900 microliters of LB bacterial culture solution, and incubate on a shaking table at 37°C for 1 hour; take an appropriate amount of culture solution Spread it on a culture plate containing appropriate antibiotics and incubate overnight in a 37°C incubator until strains grow.

[0051] 2). Expression of human GDNF fusion gene in prokaryotic cell BL21(DE3)PlysS: pick a single bacterial strain from the culture plate, grow overnight in 5 ml LB culture fluid containing appropriate antibiotics; take 500 microliters in the morning of th...

Embodiment 3

[0052] Example 3: Research on the function of human PEP-1 / GDNF expression product to penetrate the cell membrane:

[0053] 1). Preparation of PEP-1 / GDNF fusion gene expression product: After the expression bacteria are induced to express, centrifuge the expression bacteria, discard the supernatant, add serum-free eukaryotic cell culture medium to the bacteria, and fully suspend; After crushing the inclusion body and renaturation, take the supernatant of the soluble expression product, filter it through a 0.2μ filter membrane, add 10% serum and appropriate amount of antibiotics to the filtrate, and set aside;

[0054] 2). Research on the membrane penetration function of PEP-1 / GDNF fusion protein:

[0055] a. Membrane penetration test: eukaryotic Hela cells were cultured overnight, and the Hela cells were washed three times with PBS buffer solution the next day, then the above-mentioned PEP-1 / GDNF fusion protein filtrate was added, and co-cultured for 2 hours and 4 hours respect...

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Abstract

The invention discloses an expression structure method for constructing Glial cell line-Derived Neurotrophic Factor (GDNF) fusing penetrating peptides, the purification of an expressed protein product and the significance thereof, which is mainly characterized in that (1) GDNF parent is a human source mature type; (2) the N-tail end of the GDNF is fused with PEP-1 penetrating peptides comprising the 21 amino acid; and the expression product of the constructed penetrating peptides GDNF fusing the gene expression plasmid has the membrane penetrating function and biological function of GDNF and can be clinically applied in the treatment of the related diseases.

Description

technical field [0001] The invention relates to the expression plasmid construction and expression purification of human neurotrophic factor GDNF (GDNF, the same below) fused with a penetrating peptide, and its technical field is related to life science and biomedicine technology. Background technique [0002] Human glial cell-derived neurotrophic factor (GDNF, the same below) is a neurotrophic factor that is produced and secreted by glial cells in brain tissue and has important neurobiological functions. GDNF has a wide range of neurotrophic effects on the growth and differentiation of neurons on a variety of nerve cells in the central and peripheral areas. Dopamine neurons have the strongest effect (Lin LT et al. Science 1993.260(5111): 1130-2; Henderson et al. Science 1994, 266(5183): 1062-4). [0003] Extensive research on the function of GDNFF at home and abroad has proved that GDNF can significantly alleviate the chromatin condensation and DNA fragmentation induced by...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K19/00C12N15/62C12N15/63C12N1/21C12N15/86A61K38/16A61P9/10A61P25/00A61P25/16
Inventor 王尚武朱雅南
Owner 广州市凯诺生物科技有限公司
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