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A kind of recombinant type I humanized collagen polypeptide and its preparation method and application

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: 2022-06-07
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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  • A kind of recombinant type I humanized collagen polypeptide and its preparation method and application
  • A kind of recombinant type I humanized collagen polypeptide and its preparation method and application
  • A kind of recombinant type I humanized collagen polypeptide and its preparation method and application

Examples

Experimental program
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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, and the full-length is 240aa. In order to purify the TC1R4 protein after it is expressed, the present invention connects the amino acid sequence shown in SEQ ID No. 4 to the N-terminus of the amino acid sequence shown in SEQ ID No. 3 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 full length of the corresponding gene is 738 bp (refer to the underlined sequence in SEQ ID No. 6 for details). In order to facilitate the construction of the subsequent expression vector, an enzyme cleavage site sequence is added to the 5' end of th...

Embodiment 2

[0071] Example 2: Mass spectrometry 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]After DTT reduction and iodoacetamide alkylation, TC1R4 protein samples were digested with trypsin overnight. The peptides obtained after enzymatic hydrolysis were desalted with C18ZipTip, and then mixed with the matrix α-cyano-4-hydroxycinnamic acid (CHCA) to spot plate. Finally, the analysis was performed with matrix-assisted laser desorption ionization-time-of-flight mass spectrometer MALDI-TOF / TOF UltraflextremeTM, Brucker, Germany (for the technique of peptide fingerprinting, please refer to: Protein J. 201635:212-7).

[0076] Database searches were handled from the Peptide Mass Fingerprint page on the local mascot website. Protein identification results are based on primary mass spectro...

Embodiment 3

[0083] Example 3: Detection of biological activity of recombinant type I humanized collagen TC1R4

[0084] For the detection method of collagen activity, please refer to 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 ultraviolet absorption method to detect the concentration of the protein sample to be tested, including commercialized 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 (wherein the sequences of the proteins C1S4T, C1S5T refer to the patent application with the application number of 201811254050.7).

[0086] Specifically, the UV absorption of the sample at 215nm and 225nm was measured r...

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Abstract

The invention discloses a recombinant type I humanized collagen polypeptide and its preparation method and application. The recombinant type I humanized collagen polypeptide provided by the present invention comprises n repeats of the sequence shown in SEQ ID No.1, n is an integer greater than or equal to 1, wherein when n is an integer greater than or equal to 2, each repeat The sequences are directly connected; optionally, the N-terminal of the recombinant type I humanized collagen polypeptide contains an amino acid sequence that can be excised by TEV protease. The recombinant type I humanized collagen polypeptide provided by the present invention has the activity of promoting cell adhesion, the amino acid sequence of the recombinant protein is selected from the amino acid sequence of natural collagen, it will not produce an immune response when applied to the human body, and its preparation method is simple, Higher yields of collagen can be obtained at low cost.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant type I humanized collagen polypeptide and a preparation method and application thereof. Background technique [0002] Collagen (collagen) is a family of proteins, generally white, transparent, unbranched fibrils, and is the basic support of skin and bones, which can account for 25% to 35% of the total protein, mainly distributed in the human body. Skin, blood vessels, bones, tendons, teeth and cartilage are the main matrix and scaffold of these tissues, protecting and connecting various tissues and playing 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 vivo, collagen can be divided into interstitial collagen, basement membrane collagen and pericellular collagen. Interstitial collage...

Claims

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Application Information

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Patent Type & Authority Patents(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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