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A fusion protease and its preparation method, its application in extracting type I collagen and the use of the type I collagen

A collagen and protease technology, applied in the field of biogenetic engineering, can solve the problem of destroying the spatial structure of type I collagen, and achieve the effects of promoting epithelial cell proliferation, promoting collagenase production, excellent biocompatibility and biological activity

Active Publication Date: 2022-05-31
天津辅元生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the extraction efficiency of the acid enzymatic method is higher than that of the acid method, alkaline method, and enzymatic method, the enzyme in the acid enzymatic method will also degrade type I collagen and destroy the spatial structure of type I collagen while degrading miscellaneous proteins

Method used

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  • A fusion protease and its preparation method, its application in extracting type I collagen and the use of the type I collagen
  • A fusion protease and its preparation method, its application in extracting type I collagen and the use of the type I collagen
  • A fusion protease and its preparation method, its application in extracting type I collagen and the use of the type I collagen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The preparation of embodiment 1 protease

[0052] 1. Optimizing the catalytic domains of protease 7 and protease 9

[0053]On the REACTOME website, find out the protease 7 and protease 9 in the rat tail tendon of the mouse, and perform structural analysis on the protease 7 and protease 9 in the BRENDA, MEROPS, and UNIPROT databases. Find the sequence of the catalytic domain in the UNIPROT database, and optimize the sequence of the catalytic domain according to the preference of E. coli for amino acids. The sequence of the optimized protease 7 (mmp7c) is shown in the sequence table SEQ ID NO.4. The sequence of protease 9 (mmp9c) is shown in SEQ ID NO.5 of the sequence listing.

[0054] 2. Construction of recombinant plasmids and recombinant strains

[0055] The synthesized sequence and the plasmid pET21a were digested, and the sequences of mmp7c, mmp9c, mmp7C-linker-mmp9C were respectively constructed on the plasmid pET21a to obtain pET21a-mmp7c, pET21a-mmp9c and pET21...

Embodiment 2

[0106] Example 2 Detection and Analysis of Protease on Rat Tail Tendon Degradation

[0107] Divide the experiment into 10 groups, respectively numbered A1, A2, B1, B2, C1, C2, D1, D2, E1, E2, take 1g of fresh rat tail tendon, soak in 1:1000 bromogeramine solution for 10-15min After taking it out, rinse it with normal saline for more than 5 times, pour off the normal saline, and trim it to 1mm with scissors 3 The left and right tissue pieces were put into the Erlenmeyer flask, and 100ml of 0.05M Tris-Hcl (pH7.5) solution containing 1M Nacl was added, and left for 1 day. After 1 day, discard the supernatant. Add an appropriate amount of normal saline to A1 and A2, add an equal amount of acetic acid solution and trypsin solution to B1 and B2, add an equal amount of protease 7 solution to C1 and C2, and add an equal amount of protease to D1 and D2 9 solution, add an equal amount of fusion protease to E1 and E2, extract at 37°C for 8 hours, shake and observe regularly. Then cent...

Embodiment 3

[0113] Example 3 Characteristic Detection of Extracting Type I Collagen from Rat Tail Tendon Using Fusion Protease

[0114] 1. Moisture retention test: After the samples were freeze-dried, their initial moisture mass fractions were determined by the oven method. Using glycerol as a reference, freeze-dried samples were placed in a desiccator with a relative humidity of RH=81% for about 80 hours, and the relationship between their dilution rate and time was measured. Dilution rate = [(W 1 -W 0 ) / W 0 ]*100% W 0 , W 1 are the mass of the sample before placing and after placing for a certain period of time, respectively. After the moisture absorption is completed, the sample is transferred to a calcium chloride desiccator, and the relationship between the residual moisture rate of the sample and time is determined. Moisture residual rate = [(W 2 -W 0 ) / W 0 ]*100% W 0 Same as moisture absorption test; W 2 is the mass of the sample after standing for a certain period of ti...

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Abstract

The invention provides a fusion protease and its preparation method, its application in extracting type I collagen and the use of the type I collagen, the fusion protease includes protease 7 fragment mmp7C and / or protease 9 fragment mmp9C; The amino acid sequence of the protease 7 fragment mmp7C is shown in SEQ ID NO.1; the amino acid sequence of the protease 9 fragment mmp9C is shown in SEQ ID NO.2. In the process of extracting type I collagen of animal skin and tendon tissue, the fusion protease of the present invention will not destroy the spatial structure of type I collagen, and can remove miscellaneous proteins in the final product, thereby improving the extraction of type I collagen Rate.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a fusion protease and its preparation method, its application in extracting type I collagen and the use of the type I collagen. Background technique [0002] Collagen is a huge family with a wide variety and highly complex structure, with remarkable diversity from molecular structure, supramolecular structure, to tissue distribution and function, etc. They exist in various tissues of organisms, such as skin, bone, tendon, basement membrane, and the like. Type I collagen is abundant in bones and tendons. Its molecular length is about 300nm, its diameter is about 1.5nm, and it is rod-shaped. Microscopically, type I collagen consists of three peptide chains, including two α(I) chains and one α(II) chain. Because type I collagen has many biological advantages such as degradability, biocompatibility and hemostasis, it is widely used as a natural biomateri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64C12N15/62C12N15/70C12N1/21C07K14/78C07K1/14A61K8/65A61Q19/00A61Q19/08A61Q19/02A61K38/39A61P19/04A61P7/04A61P17/02C12R1/19
CPCY02A50/30
Inventor 李慧许洋
Owner 天津辅元生物医药科技有限公司
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