Polypeptide, production method and uses thereof

A sequence and region technology, applied in the field of genetic engineering, can solve the problems of long production cycle, loss of biological activity of collagen, and high cost, and achieve good hydrophilicity and stability, good cell adhesion effect, and low production cost.

Active Publication Date: 2019-02-01
SHANXI JINBO BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The collagen extracted by these methods has lost its original biological activity and cannot be applied in the field of biomedicine to exert real functions
Most of the prepared collagen products have weak tensile strength; pure collagen degrades faster in the body, and may have potential antigenicity, etc. The nutritional content and feeding value of the products are also different, and the leather is not only exposed to many chemical substances during processing, but also susceptible to bacterial infection, which severely limits the application of collagen
Although foreign research institutions have obtained milk containing human collagen by breeding mice containing human collagen genes, the cost of such production is too high and the production cycle is too long to be put into large-scale production

Method used

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  • Polypeptide, production method and uses thereof
  • Polypeptide, production method and uses thereof
  • Polypeptide, production method and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Construction and expression of recombinant human collagen polypeptide

[0069] Construction and expression of C1S4T gene expression vector

[0070] 1. The full-length gene sequence of human collagen C1S4T used in Example 1 is shown in SEQ ID No.7. This sequence has been codon optimized for E. coli codons.

[0071]2. The full length of the C1S4T gene is 747bp. According to the optimized C1S4T codon gene sequence SEQ ID No.7, Shanghai Huajin Biotechnology Co., Ltd. was entrusted to synthesize the gene fragment, and the synthesized C1S4T gene fragment was passed through BamHI (NEB Company Product number: R0136L) and Xho I (NEB company, product number: R0146L) restriction sites were inserted into PET32a expression vector. The successfully constructed expression plasmid was transformed into Escherichia coli competent cell BL21(DE3) (Merck Company). The specific process is as follows: 1: Take 1 μl of the plasmid in 100 μl of Escherichia coli competent cells BL...

Embodiment 2C1

[0085] Activity detection of embodiment 2C1S4T and C1S5T protein

[0086] 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:

[0087] 1. Use the ultraviolet absorption method to detect the concentration of the protein samples to be tested, including control human collagen (Sigma, C7774), C1S4T and C1S5T protein samples. Specifically, the ultraviolet absorption of samples at 215nm and 225nm was measured respectively, and the protein concentration was calculated by using the empirical formula C(μg / mL)=144X(A215-A225), and it should be detected under the condition of A215<1.5. The principle of this method is to measure the characteristic absorption of peptide bonds under far-ultraviolet light...

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Abstract

The present invention relates to a polypeptide, a production method and uses thereof, wherein the polypeptide comprises an N-terminal region and a C-terminal region, and has remarkable cell adhesion effect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to a polypeptide, its production method and application. Background technique: [0002] Collagen is generally white, transparent, and unbranched fibrils. It is the basic support of skin and bones and can account for 25% to 35% of the total protein. It is mainly distributed in human 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. [0003] Collagen products currently on the market are all obtained from animal tissues such as pigs, cattle, and fish. In terms of the amino acid composition of collagen: the similarity between mammalian pigs, cattle and humans is 95%, and the similarity between fish and humans is 65%. Although ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C12R1/19
CPCC07K14/78C07K2319/00C12N15/62C12N15/70C12N2800/22
Inventor 杨霞
Owner SHANXI JINBO BIO PHARMA CO LTD
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