A kind of recombinant human type xvii collagen, preparation method and application

A technology of collagen and its application, which is applied in the field of genetic engineering, can solve the problem of failure of large-scale preparation of recombinant human type XVII collagen with cell adhesion activity, and achieve optimal cell adhesion activity, low cultivation cost, tissue repair and promotion The effect of hair follicle repair and regeneration

Active Publication Date: 2021-12-14
JIANGSU TRAUTEC MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to overcome some technical problems in the prior art, mainly to overcome the optimal selection of recombinant human type XVII collagen sequence (non-unique) and whether it can be secreted and expressed efficiently in Pichia pastoris, the existing recombinant Human type XVII collagen only has cell adhesion activity, and the existing recombinant human type XVII collagen cannot be produced on a large scale.

Method used

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  • A kind of recombinant human type xvii collagen, preparation method and application
  • A kind of recombinant human type xvii collagen, preparation method and application
  • A kind of recombinant human type xvii collagen, preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0066] Example 1. Construction, Expression and Identification of Recombinant Human Type XVII Collagen

[0067] (1) Design of the amino acid sequence of recombinant human collagen type XVII

[0068] In the present invention, based on the sequence optimization of human XVII collagen, the specific sequence of the human XVII collagen is referred to: Uniprot Q9UMD9-1 sequence (https: / / www.uniprot.org / uniprot / Q9UMD9) and NCBI reference sequence Q9UMD9. 3 (https: / / www.ncbi.nlm.nih.gov / protein / Q9UMD9.3), both of which have the same sequence, as shown in SEQ ID NO.1.

[0069] SEQ ID NO. 1:

[0070]

[0071]

[0072] The bold and underlined part in the above sequence SEQ ID NO.1 is the sequence selected by the present invention, with a total of 233 amino acids. The sequence selected in the present invention is a combined sequence selected after optimization and screening from multiple helical region sequences such as the 15th helical region, the carboxyl terminal region and the ...

Embodiment 2

[0131] Example 2. Pilot-scale fermentation and protein purification of genetically engineered bacteria

[0132] (1) Pilot fermentation

[0133] The constructed engineering bacteria containing pPIC9K-170801 and pPIC9K-170802 were subjected to 50L-500L linkage pilot-scale fermentation to obtain a fermentation broth containing recombinant human type XVII collagen to realize the large-scale expression of recombinant human type XVII collagen 170801 and 170802 Production.

[0134] Seed medium YPG (formula: yeast powder 10g / L, yeast peptone FP102 20g / L, anhydrous glycerol 10g / L); fermentation medium (formula: NH 4 H 2 PO 4 190.4g / L, KH 2 PO 4 10.06g / L, CaSO 4 ·2H 2 O 1.18g / L, K 2 SO 4 18.2g / L, MgSO 4 ·7H 2 O 14.9g / L, glycerol 40g / L); feed medium (50% w / v glycerol, 12mL PTM per liter) 1 trace elements); induction medium (100% methanol with 12 mL of PTM per liter 1 trace elements); where PTM 1 Filter sterilize with a 0.22 μm filter and store at 4°C. After the fermentat...

Embodiment 3

[0155] Example 3. Detection of cell adhesion activity of recombinant collagen

[0156] References Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura. Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004) . It was commissioned by the Laboratory of Functional Nanomaterials and Biomedical Testing, School of Pharmacy, Changzhou University.

[0157] Specific implementation method:

[0158] NIH / 3T3 cells were normally cultured (purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. GNM6, and the culture and passage methods were performed according to the cell instructions). Take 170801 and 170802 protein purified freeze-dried products, and the reference substances are natural human collagen (purchased from Sigma, product number C7774) and bovine serum albumin (BSA, purchased from Sangon Bio (Shanghai) Co., Ltd.) Dissolve (ultrapure water or ...

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Abstract

The present invention provides a recombinant human type XVII collagen, which consists of or comprises the amino acid sequence shown in (A)n, wherein A is shown in SEQ ID NO.2 Sequence or a sequence after amino acid modification to a certain extent on the basis of SEQ ID NO.2 or a sequence with greater than 80% homology with SEQ ID NO.2; wherein n is an integer greater than or equal to 1; wherein each A The same or different and repeat in tandem with A as the basic unit, and each of the same or different A is directly connected by a peptide bond. In the present invention, it is verified that the collagen can not only realize the high-efficiency secretion and soluble expression of human type XVII collagen in eukaryotic host cells such as Pichia pastoris, but also has better cell adhesion than commercial natural human collagen activity, promoting cell migration, excellent tissue regeneration and biological activity promoting hair follicle repair and regeneration, and can be produced industrially.

Description

technical field [0001] The invention relates to a recombinant human type XVII collagen, a preparation method and application, and belongs to the technical field of genetic engineering. Background technique [0002] As an important natural biological protein, collagen can be widely used in many fields such as chemical industry, medicine, food, cosmetics, etc. It is especially suitable for the preparation of various biological devices. It is the most ideal source of biological materials and has broad application prospects. Collagen sold in the market is mainly the collagen extract obtained by treating animal tissues with acid, alkali and enzymatic hydrolysis. During the processing of such extracts, the natural structure of collagen is basically destroyed, the degradation is severe, and its biological activity is lost; the extracted collagen peptides have different lengths, uneven properties, unstable quality, and the risk of virus infection; at the same time, animals The amin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/78C12N15/12C12N15/81C12N1/19A61K38/39A61P17/02A61P17/14A61K8/65A61Q7/00A61L27/24A61L27/54A61L31/16A61L31/04A23L33/17C12R1/84
CPCC07K14/78C12N15/815A61P17/02A61P17/14A61K8/65A61Q7/00A61L27/24A61L27/54A61L31/16A61L31/044A23L33/17C12N2800/102C12N2800/22A61K38/00A61L2300/412A23V2002/00A23V2200/318A23V2250/5422C12R2001/84Y02A50/30A23L29/284A61K8/9728A61Q19/00A61K2800/10A61K36/06C07K2319/20C12N15/81
Inventor 李佳佳王丽萍钱松
Owner JIANGSU TRAUTEC MEDICAL TECH CO LTD
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