Recombinant human collagen and its coding gene and preparation method thereof

A technology of human collagen and human collagen, which is applied in the field of bioengineering, can solve the problems of difficulty in expressing products, difficulty in product purification, affecting product quality, etc., and achieves the effects of high expression, high purity and good water solubility.

Active Publication Date: 2017-04-05
SHAANXI HUIKANG BIO TECH CO LTD
View PDF4 Cites 23 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some research institutions and biological companies at home and abroad have successively invested in the research and development of recombinant collagen. In 2012, Yang Xia et al. achieved high expression in E. coli by repeating type III collagen peptides and fusing them with type II collagen peptides; Fan Daidi from Northwest University in my country used Escherichia coli to produce human-like collagen through high-density fermentation culture, obtained highly expressed human-like collagen, and used it as raw material to develop tissue engineering materials and c

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant human collagen and its coding gene and preparation method thereof
  • Recombinant human collagen and its coding gene and preparation method thereof
  • Recombinant human collagen and its coding gene and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0103] The preparation method of the above-mentioned gene recombinant human collagen comprises the following steps:

[0104] 1. Construction of gene recombinant human collagen expression vector

[0105] According to the amino acid sequence of the designed gene recombinant human collagen, according to the codon preference of Pichia pastoris, design and artificially synthesize the human collagen nucleotide sequence, and add the XhoI endonuclease cleavage site CTCGAG at the 5' end And Pichia pastoris signal peptide cleavage site sequence AAAAGA, 3' end added EcoRI endonuclease cleavage site GAATTC, the design sequence was synthesized by Shanghai Sangon Bioengineering Co., Ltd., named NCOL, the sequence is as follows, see the sequence list for details SEQ ID No: 3:

[0106]

[0107] XhoI and EcoRI double enzyme digestion was performed on pPIC9K and human collagen synthesized by artificial whole gene, and the digested products were recovered, ligated with DNA ligase, transforme...

Embodiment 1

[0123] 1. Construction of gene recombinant human collagen expression vector

[0124] 1.1 Gene acquisition

[0125] According to the amino acid sequence of the designed gene recombinant human collagen, according to the codon preference of Pichia pastoris, design and artificially synthesize the human collagen nucleotide sequence, and add the XhoI endonuclease cleavage site CTCGAG at the 5' end And Pichia pastoris signal peptide cleavage site sequence AAAAGA, 3' end added EcoRI endonuclease cleavage site GAATTC, the design sequence was synthesized by Shanghai Sangon Bioengineering Co., Ltd., named NCOL, the sequence is shown in the sequence table SEQ ID No: 3:

[0126] 1.2 Vector pPIC9K and NCOL double digestion

[0127] XhoI and EcoRI double enzyme digestion was performed on pPIC9K and human collagen synthesized by artificial whole gene. pPIC9K was purchased from Invitrogen Company. The double enzyme digestion system is as follows:

[0128]

[0129]

[0130] After addi...

Embodiment 2

[0155] In step 5 of the purification of genetically recombinant human collagen in Example 1, the hollow fiber microfiltration system with a pore size of 0.1 μm is replaced with a hollow fiber microfiltration system with a pore size of 0.22 μm, and other steps in this step are the same as in Embodiment 1 , the obtained product is also more than 95% genetically recombinant human collagen, its toxicity is 0, and it has good biocompatibility.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides recombinant human collagen and a method for preparing the recombinant human collagen. The method comprises the following steps: 1)preparing plasmid: performing artificial whole gene synthesis of a sequence of SEQ ID No:3 in a sequence table, then performing Xho I and EcoR I double enzyme digestion on a pPIC9K carrier and human collagen in SEQ ID No:3 through artificial whole gene synthesis, then extracting plasmid; 2) converting: mixing the prepared plasmid and pichia yeast for conversion to obtained converted bacterium colony; 3) screening of multicopy additive recombinants; 4) obtaining a broth through fermentation; and 5) purifying to obtain the product. The invention also provides a gene sequence for coding the recombinant human collagen. The recombinant human collagen has good biological compatibility, and the purity can reach more than 95%.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a gene recombinant human collagen and its coding gene, and also relates to a preparation method of the recombinant human collagen. Background technique [0002] Collagen is the most abundant protein in the body, accounting for about 25%-33% of the total protein. It is widely present in the bones, tendons, cartilage, skin and other connective tissues of the human body. It is an extracellular matrix ( ECM) plays an important role in maintaining the normal physiological functions and repairing of cells, tissues and organs. In terms of molecular structure, collagen is composed of parallel linear chains, and each linear chain is composed of three twisted left-handed α-peptide chains tightly combined through inter-chain interactions to form a very strong right-handed triple helix structure. Each α-peptide chain consists of more than 300 repeated Gly-X-Y triplets, and the two e...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C07K14/78C12N15/81C12N15/66C12N15/12C12P21/02C12N5/10
CPCC07K14/78C12N15/66C12N15/81C12N5/10C12P21/02
Inventor 侯增淼高恩李晓颖李敏杨晓琳赵金礼
Owner SHAANXI HUIKANG BIO TECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap