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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
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no technical report on the functional three-dimensional helical structure of recombinant human collagen

Method used

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  • 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

Embodiment 1

[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 Escherichia coli, and remove the NdeI and XhoI restriction sites during the design process, which is beneficial to the later stage Gene manipulation, the optimized gene sequence is shown as SEQ ID NO: 3, compar...

Embodiment 2

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

[0056] (1) Using the prokaryotic expression vector pUC-Hy726 of 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 and amplified by double digestion with Nco I and Xho I The Hy726 fragment, the gel was cut to recover Hy726 (N-X), and the results of electrophoresis separation are shown in figure 1 .

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

[0058] ddH2O 19.0 μL

[0059] 2 × pfu Buffer 25.0μL

[0060] Hy762 upstream primer 2.0 μL

[0061] Hy762 downstream primer 2.0 μL

[0062] Template 2.0μL (about 5ng)

[0063] Proceed as follows:

[0064] 94°C for 3 minutes

[0065] 94°C 30 sec

[0066] 55°C 30 sec

[0067] 72°C 1 min 40 sec

[0068] 72°C for 3 minutes

[0069] 35 cycles.

[0070] After the PCR product was purified, it was digested with N...

Embodiment 3

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

[0075] This example refers to the method of J. Sambrook et al., "Guidelines for Molecular Cloning Experiments, Third Edition", as follows: Pick a single colony of E. Add 0.5mL of overnight culture solution to a 50mL LB Erlenmeyer flask, and culture with vigorous shaking at 37°C for about 2 hours to allow the bacteria to grow to the pre-logarithmic stage; under sterile conditions, transfer the bacteria to a 50mL polypropylene tube pre-cooled with ice, Place on ice for 10min; centrifuge at 4000rpm at 4°C, pour off the supernatant, invert the tube to let the residual liquid flow out as much as possible; add 6mL of 0.1mol / L CaCl pre-cooled with ice 2 Resuspend the pellet and place it on ice for 30min; centrifuge at 3000rpm at 4°C, pour off the supernatant, invert the tube to make the residual liquid flow out as much as possible; add 1.2mL of 0.1mol / LCaCl pre-cooled with ice 2 Resuspend the pellet (if you want to ...

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PUM

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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

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Application Information

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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
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