Recombined nattokinase oral preparation, preparation method and application thereof

A technology of nattokinase and oral preparations, applied in the field of pharmacy, can solve the problem that there is no research report on oral preparations of recombinant nattokinase, and achieve the effects of uniform appearance, no adhesion and high encapsulation rate

Inactive Publication Date: 2008-12-03
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After reviewing the literature, there is currently no recombinant nattokinase oral preparation and related research reports

Method used

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  • Recombined nattokinase oral preparation, preparation method and application thereof
  • Recombined nattokinase oral preparation, preparation method and application thereof
  • Recombined nattokinase oral preparation, preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1: Cloning the full-length gene of nattokinase and constructing the expression plasmid of Bacillus subtilis

[0036] 1. Extract Bacillus natto genomic DNA (sequence 1), design cloning primers, and perform PCR with upstream primers 5'...GCGGGA TTC GTA TGA AAA TAG TTA...3' and downstream primers 5'...GTA GTC GACTCC GGT GCT TGT GAA...3' Amplify to obtain nattokinase gene and construct plasmid pBL NK.

[0037] 2. Expression of pBL NK in Bacillus subtilis expression system: Transform pBL NK plasmid into protease-deficient Bacillus subtilis by protoplast method, pick transformed colonies and inoculate them in 3ml LB culture medium containing 10μg kanamycin, shake at 34.5°C Shake for 15 hours, collect the culture solution for SDS-PAGE and activity analysis, and screen the seed engineering bacteria.

[0038]3. Purification of recombinant nattokinase: Pick freshly scratched monoclonal seed bacteria, inoculate in 3ml LB culture medium containing 10μg kanamycin, shake at ...

Embodiment 2

[0058] 1. Extract Bacillus natto genomic DNA, use upstream primer 5'...GCG GGA TTC GTA TGAAAA TAG TTA...3' and downstream primer 5'...GTA GTC GAC TCC GGT GCT TGTGAA...3' for PCR amplification to obtain nattokinase gene , construction of plasmid pBL NK

[0059] 2. Expression of pBL NK in Bacillus subtilis expression system: Transform pBL NK plasmid into protease-deficient Bacillus subtilis by protoplast method, pick transformed colonies and inoculate them in 3ml LB culture medium containing 10μg kanamycin, shake at 34.5°C Shake for 15 hours, collect the culture solution for SDS-PAGE and activity analysis, and screen the seed engineering bacteria.

[0060] 3. Purification of recombinant nattokinase: Pick freshly scratched monoclonal seed bacteria, inoculate in 3ml LB culture medium containing 10μg kanamycin, shake at 37°C for 12h, inoculate in 1L culture medium at 1:1000 Shake at 34.5°C for 15 hours, centrifuge at 8000rpm and collect the supernatant. 1L of the supernatant is ul...

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Abstract

The invention belongs to the pharmacy field, particularly relating to a method for preparing a recombinant nattokinase specific oral preparation and an application thereof. The invention utilizes a supercritical CO2 fluid coating technology and takes methacrylate- ethylacrylate copolymer (Eudragit L100-55) as coating materials to coat recombinant nattokinase medicine particles to prepare the oral preparation. The particles of the recombinant nattokinase preparation have the advantages of regularity, smooth surface, uniform appearance, no adhesion and high drug-loading amount and entrapment rate, and the average particle diameter is between 105 and 165 mu m. The test results show that the method realizes the resistance of gastric acids and the slow release reaction of the recombinant nattokinase oral preparation.

Description

technical field [0001] The invention belongs to the field of pharmacy, and in particular relates to a recombinant nattokinase oral preparation and a preparation method and application thereof. Background technique [0002] Recombinant nattokinase is a protein / polypeptide drug, which is directly taken orally, and is easily destroyed by gastric acid and gastrointestinal protease to lose its biological activity, so its clinical application is limited. The gastrointestinal tract constitutes three major barriers to the oral absorption of protein / peptide drugs: enzymatic degradation, the diffusion barrier of the mucus layer, and the absorption barrier of the mucous membrane. The conventional preparation process requires the use of high temperature, water, and organic solvents, which can easily cause protein degradation or loss of activity, so that it is very difficult to make protein / polypeptide drugs into oral preparations. However, in recent years, oral colon-targeted drug deliv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/48A61K9/16A61K47/32A61P1/04
Inventor 宋后燕莫炜马晓文
Owner FUDAN UNIV
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