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Recombinant I-type humanized collagen polypeptide as well as preparation method and application thereof

A collagen polypeptide and humanized technology, applied in the field of genetic engineering, can solve the problems of long cycle time, high production cost of human collagen, and inability to produce large-scale production, and achieve the effect of simple preparation method and good cell adhesion effect

Active Publication Date: 2021-11-09
SHANXI JINBO BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the low bioactivity of heterologous collagen in this field, easy to induce immune response, and the low utilization rate of collagen caused by a series of problems such as high production cost, long cycle, and inability to produce large-scale production of human collagen, this The invention provides a recombinant type I humanized collagen polypeptide, as well as its preparation method and use

Method used

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  • Recombinant I-type humanized collagen polypeptide as well as preparation method and application thereof
  • Recombinant I-type humanized collagen polypeptide as well as preparation method and application thereof
  • Recombinant I-type humanized collagen polypeptide as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Preparation of recombinant type I humanized collagen TC1R4

[0058] 1. Construction of TC1R4 gene expression vector

[0059] The full-length protein sequence of the recombinant type I humanized collagen TC1R4 used in this example is the amino acid sequence shown in SEQ ID No.3, with a full length of 240 aa. In order to purify the TC1R4 protein after it is expressed, the N-terminal of the amino acid sequence shown in SEQ ID No.3 is connected with the amino acid sequence shown in SEQ ID No.4 to form the amino acid sequence shown in SEQ ID No.5 , its full length is 246aa. The coding sequence corresponding to the amino acid sequence was codon-optimized for the codons of Escherichia coli, and the corresponding gene has a full length of 738 bp (see the underlined sequence in SEQ ID No. 6 for details). In order to facilitate the construction of subsequent expression vectors, a restriction site sequence was added to the 5' end of the coding sequence, and a stop cod...

Embodiment 2

[0071] Example 2: Mass Spectrometric Detection of Recombinant Type I Humanized Collagen TC1R4

[0072] Mass spectrometry detection of TC1R4 protein was performed according to the conditions shown in Table 1.

[0073] Table 1 Conditions for mass spectrometry detection

[0074]

[0075]TC1R4 protein samples were treated with DTT reduction and iodoacetamide alkylation, and then digested with trypsin overnight. The peptides obtained after enzymatic hydrolysis were desalted by C18ZipTip, mixed with matrix α-cyano-4-hydroxycinnamic acid (CHCA) and plated. Finally, matrix-assisted laser desorption ionization-time-of-flight mass spectrometer MALDI-TOF / TOFUltraflextremeTM, Brucker, Germany was used for analysis (for the technique of peptide fingerprinting, please refer to: ProteinJ.201635:212-7).

[0076] Database retrieval was handled from the Peptide Mass Fingerprint page on the local mascot website. The protein identification results were obtained based on the primary mass spe...

Embodiment 3

[0083] Example 3: Bioactivity Detection of Recombinant Type I Humanized Collagen TC1R4

[0084] Collagen activity detection method can refer to the literature 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). The specific implementation method is as follows:

[0085] (1) Use the ultraviolet absorption method to detect the concentration of the protein sample to be tested, including commercial human collagen (Sigma, C7774), recombinant type I humanized collagen TC1R4 provided by the present invention, recombinant type III collagen C1S4T, recombinant Type III collagen C1S5T (for the sequences of the proteins C1S4T and C1S5T, please refer to the patent application with application number 201811254050.7).

[0086] Specifically, the UV absorption of samples at 215nm and 225nm was measured respectively, an...

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Abstract

The invention discloses a recombinant I-type humanized collagen polypeptide as well as a preparation method and application thereof. The recombinant I-type humanized collagen polypeptide provided by the invention comprises n repeats of a sequence shown in SEQ ID No. 1, wherein n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repeat sequences are directly connected; and optionally, the N tail end of the recombinant I-type humanized collagen polypeptide comprises an amino acid sequence which can be excised by TEV protease. The recombinant I-type humanized collagen polypeptide provided by the invention has the activity of promoting cell adhesion, the amino acid sequence of the recombinant protein is selected from a natural collagen amino acid sequence, and the recombinant protein does not generate an immune response when being applied to a human body; and moreover, the preparation method is simple, and high-yield collagen can be obtained at low cost.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant type I humanized collagen polypeptide and its preparation method and application. Background technique [0002] Collagen is a kind of protein family, generally white, transparent, unbranched fibrils, is the basic support of skin and bones, can account for 25% to 35% of the total protein, and is mainly distributed in the human body. Skin, blood vessels, bones, tendons, teeth, and cartilage are the main matrix and scaffolding of these tissues, protecting and connecting various tissues, and performing important physiological functions in the body. Therefore, collagen can be widely used in industries such as medicine and cosmetics. According to the distribution and functional characteristics of collagen in the body, collagen can be divided into interstitial collagen, basement membrane collagen and peripheral collagen. Interstitial collagen molec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C12N15/12C12N15/70C12N1/21C07K1/18C07K19/00C12N15/62A23L33/18A61K38/39A61P43/00A61K8/65A61Q19/00
CPCC07K14/78C12N15/70A23L33/18A61P43/00A61K8/65A61Q19/00C12N2800/22C07K2319/50A23V2002/00A61K38/00A61K2800/10A23V2200/30A23V2250/55
Inventor 杨霞陆晨阳兰小宾何振瑞王建王玲玲
Owner SHANXI JINBO BIO PHARMA CO LTD
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