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Recombinant human keratinocyte factor KGF-2 (keratinocyte growth factor-2) environment-sensitive eye transfer system and application thereof

A KGF-2, environment-sensitive technology, applied in the direction of digestive system, respiratory system diseases, sensory diseases, etc., can solve problems such as expensive products, affecting widespread use, heat and acid instability, etc.

Active Publication Date: 2014-03-26
广州力鑫生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are two main defects in the biological preparation of the KGF-2 protein: (1) the general expression level of KGF-2 is low (with E. The large-scale production of the drug has caused the price of the product to be very expensive, which has affected its widespread use; (2) KGF-2, like other protein drugs, is unstable to heat and acid, and its half-life in the body is only a few minutes. Systemic application It will be quickly inactivated and it will be difficult to play its effective role. Local puncture administration will damage the deep nerves and it is difficult to ensure the accuracy of each drug release site, and it is not suitable for long-term medication.
However, while bFGF and EGF promote the healing of corneal epithelial injuries, they also promote the growth of new blood vessels, leaving thin nebula, nebula or white spots after healing, which have different degrees of impact on vision
Although keratocytes can secrete a certain amount of KGF-2, exogenous KGF-2 is still needed for timely repair to promote the proliferation and migration of stromal cells

Method used

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  • Recombinant human keratinocyte factor KGF-2 (keratinocyte growth factor-2) environment-sensitive eye transfer system and application thereof
  • Recombinant human keratinocyte factor KGF-2 (keratinocyte growth factor-2) environment-sensitive eye transfer system and application thereof
  • Recombinant human keratinocyte factor KGF-2 (keratinocyte growth factor-2) environment-sensitive eye transfer system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1. Expression and purification of recombinant human KGF-2 protein

[0086] Adopt SUMO fusion protein expression system, use Escherichia coli to express the host, use LB culture medium to ferment, and the cell broken liquid is separated by cation exchange chromatography, heparin affinity chromatography column and HPLC, and the purified recombinant human KGF-2 protein is collected. . The amino acid sequence of the recombinant human KGF-2 protein is shown in SEQ ID No.1.

[0087] Firstly, the molecular chaperone SUMO is used as the fusion tag for expressing rhKGF-2. On the one hand, this tag can improve the solubility of the target protein and help it fold correctly, so as to achieve the purpose of obtaining a large amount of soluble fusion protein, and it can also greatly improve the rhKGF-2. The expression rate is favorable for large-scale fermentation production. For details, please refer to the literature (relevant literature is provided) (Molecular Biotec...

Embodiment 2

[0088] Embodiment two, the preparation of recombinant human KGF-2 siliceous body

[0089] (a) Weigh 3 mg of organic-inorganic composite lipids selected from any one of formulas 1-4, acidify them in ethanol hydrochloric acid solution with pH=3, place them in a 25 mL round bottom flask, spin dry, and dissolve them in an appropriate amount of chloroform , and then slowly rotary evaporated (55 ° C water bath, 90r / min) to remove the solvent, and then rotated for 30 min after the solvent evaporated to fully remove traces of chloroform;

[0090] (b) Add ultrapure water with a total volume of 4-5 mL, an aqueous solution of recombinant human KGF-2 (the molar ratio of the complex lipid to recombinant human KGF-2 is 10:1) and alkyl glucoside (alkyl Glucoside is used as a protein protectant, wherein the molar ratio of the alkyl glucoside to recombinant human KGF-2 is 3:1), the flask is placed in a constant temperature water bath at 50°C to fully hydrate the film for 30min, and then the wa...

Embodiment 3

[0092] Embodiment three, the preparation of recombinant human KGF-2 siliceous body

[0093] (a) Weigh 3 mg of organic-inorganic composite lipids selected from any one of formulas 1-4, acidify them in ethanol hydrochloric acid solution with pH=3, place them in a 25 mL round bottom flask, spin dry, and dissolve them in an appropriate amount of chloroform , and then slowly rotary evaporated (55 ° C water bath, 90r / min) to remove the solvent, and then rotated for 30 min after the solvent evaporated to fully remove traces of chloroform;

[0094] (b) The template microsphere solution (i.e., SiO 2 Particles) were added to the lipid film, and after incubation for 3 hours, ultrasonication was carried out in a water bath;

[0095] (c) leave the solution obtained after ultrasonication at room temperature, and after the inorganic silicate shell is formed on the surface of the template microspheres, remove the template microspheres by physical or chemical methods to obtain a blank siliceo...

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Abstract

The invention discloses a recombinant human keratinocyte factor KGF-2 (keratinocyte growth factor-2) environment-sensitive eye transfer system and application thereof, and particularly relates to an environment-sensitive eye transfer system taking KGF-2 silicon plastid as a core and a preparation method and application thereof, and the transfer system comprises the following components by weight: 1.2-3.5 wt% of KFG-2, 1-10wt% of organic - inorganic compound lipid, 6-20wt% of a protective agent and 1-5wt% of substrate gel. The KGF-2 silicon plastid is prepared by thin film hydration ultrasound, and further the environment sensitive eye transfer system is prepared. Compared with the prior art, the product can obviously improve the stability of the KGF-2 and prolong the retention time of the KGF-2 in the eye, is conducive to the absorption and utilization of the KGF-2, has good repairing effects on various corneal ulcers, also can moisten the eyeball and alleviate the dry eye symptoms, has good relieving effects on eye fatigues, and also is suitable for the repairing of other oral mucosas and mucosa trauma and the repairing after skin beauty treatment.

Description

technical field [0001] The invention belongs to the field of genetic engineering pharmaceutical preparations, and discloses a recombinant human keratinocyte factor KGF-2 environment-sensitive ocular delivery system and application thereof. It specifically relates to an environment-sensitive eye delivery system with KGF-2 siliceous body as the core, its preparation method and application. The KGF-2 siliceous body is prepared by a film hydration ultrasonic method, and further prepared into an environment-sensitive ocular delivery system. Background technique [0002] Keratinocyte growth factor-2 (KGF-2) is the tenth member of the fibroblast growth factor family and is a polypeptide growth factor that exists in the body itself. KGF-2 is a single-copy gene consisting of 3 exons and 2 Composed of two introns, located on chromosome 15, it is a single-chain polypeptide containing 5 cysteine ​​residues, of which 4 cysteines form 2 pairs of disulfide bonds, and the other cysteine ​​...

Claims

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Application Information

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IPC IPC(8): A61K38/18A61K9/127A61K8/64A61K8/14A61K47/24A61P27/02A61P1/02A61P11/02A61Q19/00
Inventor 黄亚东曲红艳项琪苏志坚李校堃王晓杰
Owner 广州力鑫生物科技有限公司
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