Recombinant humanized III-type collagen and preparation method thereof

A technology of human collagen and production method, which is applied in the field of recombinant type III human collagen and its production, and can solve problems such as inability to perform biological functions, human immune rejection, and hidden dangers of viruses

Pending Publication Date: 2021-01-08
叶华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional way to obtain collagen is to extract it from animals. The process involves acid-base enzymatic hydrolysis, which can easily cause collagen to lose its original physiological a

Method used

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  • Recombinant humanized III-type collagen and preparation method thereof
  • Recombinant humanized III-type collagen and preparation method thereof
  • Recombinant humanized III-type collagen and preparation method thereof

Examples

Experimental program
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Embodiment 1

[0040] Construction of co-expression vector of human collagen type III gene and PBCV-1 proline hydroxylase:

[0041]Add BglII and SacI restriction sites to the 5' and 3' ends of the optimized human collagen type III gene, synthesize the gene, transform and extract the plasmid vector where a large number of genes are located, digest with BglII and SacI, and agarose gel Recovery and purification. The pESC-URA plasmid vector was digested and purified by the same method. The human collagen type III gene fragment and the pESC-URA plasmid vector fragment were ligated with T4 DNA ligase overnight at 16°C, transformed into Escherichia coli DH5α, the clones were screened on the ampicillin LB plate, and the plasmid was extracted and identified by double enzyme digestion (ClaI— SacI), select the successful single clone, expand the culture and extract the plasmid as a vector, digest it with BamHI and SalI, recover and purify it on agarose gel. Add BamHI and SalI restriction sites to the...

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Abstract

The invention provides a recombinant humanized III-type collagen. The recombinant humanized III-type collagen has a triple helix structure, and has an amino acid sequence reducing a humanized III-typecollagen alpha-1 chain and including 1068 amino acids. The invention further provides a preparation method of the recombinant humanized III-type collagen, wherein the prolyl hydroxylase of PBCV-1 includes 209 amino acids, and the base sequence is optimized according to the codons of saccharomyces cerevisiae. Compared with the prior art, the preparation method has the advantages that the triple helix is formed in vitro from collagen chains by utilizing the prolyl hydroxylase; and the saccharomyces cerevisiae expression system adopted by the invention is applicable to large-scale amplificationwithout endotoxin or later removal, the preparation cost is low, and the protein expression quantity is further improved. The recombinant humanized III-type collagen is excellent in stability, the amino acid composition of the recombinant humanized III-type collagen is almost completely the same as that of the alpha-1 chain of a natural collagen, and no immunological rejection can be generated when the recombinant humanized III-type collagen is applied to a human body. The recombinant humanized III-type collagen is good in biocompatibility and can be widely applied to surgical treatment and cosmetic medical industry.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a recombinant type III human collagen and a production method thereof. Background technique [0002] Collagen is an important structural protein that widely exists in the connective tissue of the human body. It accounts for about 1 / 3 of the total protein of the human body and 3 / 4 of the dry weight of the skin. It acts as a scaffold in the cytoplasmic matrix to keep the skin elastic. Type III collagen mainly exists in blood vessels, skin and intestinal tract, and its loss is the main cause of skin aging. Therefore, in the current medical aesthetics industry, type III collagen is the main research object. [0003] The traditional way to obtain collagen is to extract it from animals. The treatment process involves acid-base enzymatic hydrolysis, which tends to cause collagen to lose its original physiological activity and fail to perform its normal biological functions. ...

Claims

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Application Information

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IPC IPC(8): C07K14/78C12N15/81C12N9/02C12R1/865
CPCC07K14/78C12N15/81C12N9/0071C12Y114/11002C07K2299/00C12N2800/22
Inventor 叶华
Owner 叶华
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