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Liposome-encapsulated recombinant human ciliary neurotrophic factor

A ciliary neurotrophic and liposome technology, which is applied in liposome delivery, nervous system diseases, drug combination, etc., can solve the problems of clinical application impact, CNTF protein toxicity and side effects, etc.

Inactive Publication Date: 2012-05-23
CHONGQING FAGEN BIOMEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high toxicity and side effects of the natural sequence CNTF protein, its clinical application is affected

Method used

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  • Liposome-encapsulated recombinant human ciliary neurotrophic factor
  • Liposome-encapsulated recombinant human ciliary neurotrophic factor
  • Liposome-encapsulated recombinant human ciliary neurotrophic factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1rhCNTF (A 17 R 63 15) Acquisition of genes

[0014] According to the CNTF cDNA sequence obtained from Gene Bank, 15 amino acids were removed from the C-terminal of the cDNA of wild-type human CNTF to increase its biological activity. At the same time, Cys at position 17 was mutated to Ala, Gln at position 63 was mutated to Arg, and a band was designed on the gene sequence. There are NdeI restriction sites and ATG translation start codons, and the downstream primers have BamHI restriction sites and TAA stop codons, all using E. coli preferred codons, the whole gene is artificially synthesized, embedded in pUC18 vector and named pUC-CNTF. Corresponding base and amino acid sequence analysis such as figure 1

Embodiment 2

[0015] Construction and expression of embodiment 2 plasmid pET-CNTF

[0016] After double digestion with Nde I and BamHI, ligate with the pET-3c large fragment digested with Nde I and BamHI under the action of T4 ligase, transform Escherichia coli DH5α and coat LB-Ampicillin plates, extract positive clone plasmids, and use Nde I and BamHI double enzyme digestion identified correctly, named pET-CNTF. The recombinant expression plasmid is transformed into BL21(DE3) plyss to obtain engineering bacteria recombinantly expressing CNTF. IPTG-induced expression screened high-expressing strains and saved them. The sequencing results of engineering bacteria are as follows: figure 2 . Engineering bacteria induced expression such as image 3 a.

Embodiment 3

[0017] The separation and purification of embodiment 3 rhCNTF

[0018] Experiments proved that rhCNTF recombinant protein was expressed as inclusion body.

[0019] 1. Put the bacteria stored in the refrigerator into the bacterial lysate at a ratio of 1:10, stir at 37°C for 4 hours, and then sonicate. After cooling at 4°C, press the power P<400W for 5 seconds, with an interval of 5 seconds and sonicate 40 times, 12000r Centrifuge for 10 min at 1 / min, take the supernatant for electrophoresis, and further process the inclusion bodies.

[0020] 2. Put the inclusion body into washing solution A (20mmol / L Tris-HCl pH 8.0, 5mmol / L EDTA, 2mol / L Urea, 1% TritonX-100), ultrasonic power P<340W, working time 5 seconds, intermittent 5 seconds Ultrasound 40 times per second. After stirring for 30 minutes, centrifuge at 12,000 r / min for 15 minutes, and take 60 μL of supernatant A for SDS-PAGE.

[0021] 3. Put the inclusion body into the washing solution B (20mmol / L Tris-HCl pH 8.0, 4mol / L...

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Abstract

The invention provides a liposome-encapsulated recombinant human ciliary neurotrophic factor (rhCNTF) containing monosialo ganglioside (GM1) and a preparation method thereof. Particle diameters of liposome of the rhCNTF prepared by adopting the preparation method is evenly distributed within a range of 20 to 200nm, and the drug encapsulating rate is no less than 70 percent. An animal experiment proves that by adopting the rhCNTF liposome, the rhCNTF can pass through a blood brain barrier, and the rhCNTF liposome has the application value of curing diseases related to neuronal damages and degenerative diseases.

Description

technical field [0001] The invention relates to a preparation method of liposome containing recombinant human ciliary neurotrophic factor (rhCNTF), liposome formula and application. Background technique [0002] Ciliary Neurotrophic Factor (Ciliary Neurotrophic Factor, CNTF) was originally extracted from the ciliary ganglion of the ocular tissue of the chicken embryo. It has been found through research that CNTF has multiple functions, is of great significance in the treatment of nervous system, muscular system, obesity and related diseases, and has broad prospects for clinical development. However, due to the obstruction of the blood-brain barrier, CNTF is limited in reaching the central nervous system, which hinders the use of CNTF in the treatment of Parkinson's disease, Alzheimer's disease and other diseases. The liposome complex preparation can help drug molecules to cross the blood-brain barrier. CNTF provides a feasible way to drug in the central nervous system. Bec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K9/127A61K47/28A61K38/18A61P25/00
Inventor 高剑坤杨黎张继兰陈海容胡春兰范开
Owner CHONGQING FAGEN BIOMEDICAL
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