Recombinant human type III collagen having functional structure and expression method thereof

A collagen, functional structure technology, applied in the direction of animal/human protein, microbial-based methods, connective tissue peptides, etc., to achieve good biological activity and collagen stability

Active Publication Date: 2020-05-01
河北纳科生物科技有限公司
View PDF10 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no technical report on the functional three-dim

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 type III collagen having functional structure and expression method thereof
  • Recombinant human type III collagen having functional structure and expression method thereof
  • Recombinant human type III collagen having functional structure and expression method thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0041] Example 1 Gene design and synthesis

[0042] (1) Genetic design:

[0043] According to the amino acid sequence of human type III collagen (UniProtKB / Swiss-Prot: P02461.4), SEQ ID NO:1:

[0044] MYDSYDVKSGVAVGGLAGYPGPPGPPPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESPGGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAGIGGEKAGGFAPYYHHHHHH

[0045] Use the online design tool Jcat (http: / / www.jcat.de / ) to reverse design the gene sequence, aiming at the preferred codons required for expression in the host E. coli, and remove the NdeI and XhoI restriction sites during the design process, which is advantageous for the later stage Genetic manipulation, the optimized gene sequence is shown in SEQ ID NO: 3. Compared with the o...

Example Embodiment

[0055] Example 2 Construction of expression vector pACYCDuet 1-Hy726-1230

[0056] (1) Using the prokaryotic expression vector pUC-Hy726 of the hydroxylase Hy726 obtained in Example 1 as a template, the Hy726 fragment was amplified and cloned by high-fidelity PCR technology, and the PCR product was purified by double enzyme digestion with Nco I and Xho I. Hy726 fragments, cut the gel to recover Hy726 (NX), electrophoresis separation results see figure 1 .

[0057] The PCR amplification system is as follows (take 50μL as an example):

[0058] ddH2O 19.0μL

[0059] 2 × pfu Buffer 25.0μL

[0060] Hy762 upstream primer 2.0μL

[0061] Hy762 downstream primer 2.0μL

[0062] Template 2.0μL (about 5ng)

[0063] Proceed as follows:

[0064] 94℃ 3 min

[0065] 94℃ 30 sec

[0066] 55℃ 30 sec

[0067] 72℃ 1 min 40 sec

[0068] 72℃ 3 min

[0069] 35 cycles.

[0070] After the PCR product was purified, it was digested with Nco I and BamH I, and the digested product was digested and recovered (the band size w...

Example Embodiment

[0074] Example 3 Construction of BL21(DE3) / pACYCDuet 1-Hy726-1230

[0075] This example refers to the method of J. Sambrook et al., "Molecular Cloning Test Guide Third Edition" method), and the details are as follows: pick a single colony of E. coli BL21 (DE3) into an LB test tube and shake it overnight at 37°C; Add 0.5mL of overnight culture solution to a 50mL LB Erlenmeyer flask, culture with vigorous shaking at 37°C for about 2 hours to make the bacteria grow to the early logarithmic stage; under aseptic conditions transfer the bacteria to a 50mL polypropylene tube pre-cooled with ice, Place on ice for 10 minutes; centrifuge at 4°C at 4000 rpm, pour out the supernatant, invert the tube to make the residual liquid flow out as much as possible; add 6 mL of 0.1mol / L CaCl pre-cooled with ice 2 Resuspend the pellet and place it on ice for 30 minutes; centrifuge at 4°C at 3000 rpm, pour out the supernatant, invert the tube to make the residual liquid flow out as much as possible; a...

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 relates to a recombinant human type III collagen having a functional structure and a prokaryotic expression method thereof. The nucleotide sequence of an encoding gene of the human typeIII collagen is shown in SEQ ID NO:3, and through coexpression with hydroxylase Hy726 with a gene sequence shown in SEQ ID NO:4, the recombinant human type III collagen with the tertiary helical structure is obtained. The protein has better stability and functionality, and the biological activity is more comparable to biological activity of a natural human type III collagen.

Description

technical field [0001] The invention belongs to the technical field of optimized coding genes, and in particular relates to a recombinant human type III collagen with functional structure and a prokaryotic expression method thereof. Background technique [0002] Body collagen, also known as collagen, is a glycoprotein, and type III collagen is a triple helix formed by three peptide chains twisted to the right. The primary structure analysis showed that the long segment sequence of its polypeptide chain was formed by repeating GLy-x-y amino acid sequence. Among them, x is usually proline, y is usually hydroxyproline and hydroxylysine, this tripeptide repeat sequence plays a great role in the structure of collagen, especially the collagen domain of fibroblast collagen is composed of A long uninterrupted triple helix. Hydroxyproline and hydroxylysine are rare in other proteins. The hydroxyl group of hydroxyproline can participate in the formation of hydrogen bonds between cha...

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): C07K14/78C12N15/12C12N15/70C12N15/66C12N9/02C12N15/53C12N1/21C12R1/19
CPCC07K14/78C12N9/0071C12N15/66C12N15/70
Inventor 徐兰举齐磊刘鑫申翠美杜亚东
Owner 河北纳科生物科技有限公司
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