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Recombinant III-type humanized collagen, nucleic acid, carrier and implant

A human collagen and collagen technology, applied in the field of genetic engineering, can solve the problems of easy degradation, poor stability of recombinant humanized collagen, limited application, etc., and achieve the effect of high stability

Active Publication Date: 2022-02-25
西安德诺海思医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the existing technical defects and provide a recombinant type III humanized collagen, nucleic acid, carrier and implant, so as to solve the problem of the preservation and use of recombinant humanized collagen in liquid dosage form in the prior art. Poor stability in the process, easy to degrade, and limit its clinical application

Method used

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  • Recombinant III-type humanized collagen, nucleic acid, carrier and implant
  • Recombinant III-type humanized collagen, nucleic acid, carrier and implant
  • Recombinant III-type humanized collagen, nucleic acid, carrier and implant

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Experimental program
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Effect test

Embodiment 1

[0044] (1) Structural Design of Recombinant Type III Humanized Collagen

[0045] According to the present invention, four peptide chains of natural human type III collagen α1 chain sequence (reference sequence Genebank accession number: NM_000090.3) 549-560aa, 597-613aa, 881-902aa, 648-662aa are spliced ​​in order to form a monomer, See SEQ.ID.NO.5 for the amino acid sequence of the monomer.

[0046] In the present invention, the monomers composed of the above-mentioned tetrapeptide segments are used for repetitive tandem expression.

[0047] (2) Preparation of recombinant type III humanized collagen

[0048] 2.1 Acquisition of genes

[0049] The amino acid sequence of the above monomer is designed according to the codon preference of Pichia pastoris, and the corresponding nucleotide sequence is designed. At the same time, for subsequent molecular operations, the DNA sequence is added with Xho I restriction endonuclease sites CTCGAG and KEX2 at the 5' end Enzyme cutting sit...

Embodiment 2

[0057] Preparation of Monomeric Ten-repeat Recombinant Type III Humanized Collagen

[0058] (1) Construction of genetically engineered bacteria

[0059] After extracting the multi-repeat tandem expression plasmid obtained in Example 1, preferably the ten-repeat tandem pPIC9K-COL3-10 plasmid, linearize it with restriction endonuclease Sal I, and then transform Pichia pastoris by electroporation Host strain GS115 (Invitrogen), high-copy transformants were screened by G418 (4mg / mL, YPD medium), and the expression was confirmed by shake flask fermentation (29°C, 200rpm, BMMY medium, methanol feed 1% at intervals of 24 hours) Happening. The shake flask fermentation supernatant was detected by SDS-PAGE electrophoresis, from figure 1 It can be seen from the figure that there is an obvious electrophoresis band at about 60KD, and it is confirmed that the recombinant type III humanized collagen genetically engineered bacteria with monomer ten repeats was obtained.

[0060] (2) Fermen...

Embodiment 3

[0063] The obtained recombinant type III humanized collagen freeze-dried powder is prepared into a 0.1% liquid with purified water, sealed and subpackaged after sterile filtration, and placed under the conditions of a temperature of 40°C ± 2°C and a relative humidity of 75% ± 5%. Accelerated experiments were carried out at room temperature at 25°C±2°C, relative humidity 60%±5%, samples were taken at different time points, and the integrity of the recombinant type III humanized collagen was detected by SDS-PAGE electrophoresis. Test results such as Figure 4 shown; from Figure 4 It can be seen from the figure that at room temperature of 25°C, when samples were taken at 3 and 6 months, the protein bands were complete and there was almost no degradation; at accelerated conditions at 40°C, when samples were taken at 3 and 6 months, at Complete, almost no degradation, sampling in June, a little protein degradation. From the above experimental results, it can be concluded that th...

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Abstract

The invention discloses a recombinant III-type humanized collagen, a nucleic acid, a carrier and an implant, and relates to the technical field of biological genes. Sequence peptide fragments 549-560aa, 597-613aa, 881-902aa and 648-662aa which are obtained by screening a natural human III type collagen alpha1 chain for a long time and have good stability are selected, the four peptide fragments are spliced in sequence to form monomers, and then tandem expression is performed on the monomers. Through fermentation and purification, the obtained recombinant III-type humanized collagen is high in stability, the problem of degradation of the recombinant III-type humanized collagen in the clinical use process is effectively solved, and the recombinant III-type humanized collagen can be prepared into a liquid preparation for preservation and use.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant type III humanized collagen, nucleic acid, carrier and implant. Background technique [0002] Collagen is the most abundant protein in the body and the main component of the extracellular matrix (ECM). It plays an important role in maintaining the normal physiological functions and repairing damage of cells, tissues and organs. It has been widely used in medicine, Health care products and cosmetics industry. In terms of molecular structure, each collagen peptide chain is mainly composed of Gly-X-Y structural peptide chains (X, Y are any amino acid residues other than Gly), this peptide chain structure is necessary for the formation of collagen fiber advanced structure, determines Collagen has excellent biocompatibility and low immunogenicity. [0003] The main source of collagen is extracted from animal tissues, and with the development of biotechnolog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C12N15/81C12P21/02C07K1/34C12N15/12A61L27/26A61L27/60C12R1/84
CPCC07K14/78C12N15/815A61L27/26A61L27/60C12N2800/22C08L89/00C08L5/08
Inventor 侯增淼魏文培周浩郝东赵硕文
Owner 西安德诺海思医疗科技有限公司
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