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Recombinant human XVII type collagen, and preparation method and application thereof

A collagen and application technology, applied in the field of genetic engineering, can solve the problem of cell adhesion active recombinant human type XVII collagen failing to be produced on a large scale

Active Publication Date: 2021-07-30
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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  • Recombinant human XVII type collagen, and preparation method and application thereof
  • Recombinant human XVII type collagen, and preparation method and application thereof
  • Recombinant human XVII type collagen, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Construction, expression and identification of recombinant human type XVII collagen

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

[0068] In the present invention, based on the optimization of human type XVII collagen sequence, the specific sequence reference of human type XVII collagen is: 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), the two sequences are identical, as shown in SEQ ID NO.1.

[0069] SEQ ID NO.1:

[0070]

[0071]

[0072] The bold underlined part of the above sequence SEQ ID NO.1 is the sequence selected in 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 middle re...

Embodiment 2

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

[0132] (1) Pilot fermentation

[0133] The engineered bacteria containing pPIC9K-170801 and pPIC9K-170802 respectively were fermented in a 50L-500L linkage pilot test to obtain a fermentation broth containing recombinant human type XVII collagen to achieve large-scale expression of recombinant human type XVII collagen 170801 and 170802 Production.

[0134] Seed medium YPG (recipe: yeast powder 10g / L, yeast peptone FP102 20g / L, anhydrous glycerin 10g / L); fermentation medium (recipe: 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, add 12mL PTM per liter 1 trace elements); induction medium (100% methanol, 12 mL PTM per liter 1 trace elements); where PTM 1 Sterilize by filtration with a 0.22 μm membrane filter and store at 4°C. After the fermentation med...

Embodiment 3

[0155] Example 3. Cell adhesion activity detection of recombinant collagen

[0156] References for Cell Adhesion Detection Method of Recombinant Collagen 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) . Entrusted to the Functional Nanomaterials and Biomedical Testing Laboratory of Changzhou University School of Pharmacy to complete.

[0157] Specific implementation method:

[0158] NIH / 3T3 cells were cultured normally (purchased from the Cell Bank of the Chinese Academy of Sciences, Cat. No. GNM6, and the cultivation and passage methods were performed according to the instructions of the cells). Take 170801 and 170802 protein purification and freeze-dried products, and the reference substance is natural human collagen (purchased from Sigma, item number C7774) and bovine serum albumin (BSA, purchase...

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Abstract

The invention provides a recombinant human XVII type collagen. The collagen is composed of amino acid sequence shown as (A)n or comprises an amino acid sequence shown as (A)n, wherein A is a sequence shown as SEQ ID NO.2 or a sequence obtained by carrying out amino acid modification to a certain extent on the basis of SEQ ID NO.2 or a sequence having more than 80% of homology with SEQ ID NO.2; n is an integer greater than or equal to 1; and each A is the same or different and is serially repeated by taking A as a basic unit, and each same or different A is directly connected through a peptide bond. It is verified that the collagen not only can realize the efficient secretion soluble expression of the human XVII type collagen in the pichia pastoris which is an eukaryotic host cell, but also can realize the high-efficiency secretion soluble expression of the human XVII type collagen; in addition, the collagen also has more excellent cell adhesion activity, cell migration promotion activity and excellent tissue regeneration and biological activity of promoting hair follicle repair and regeneration than commercial natural human collagen, and can be industrially produced.

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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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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