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Recombinant humanized III type collagen alpha 1 chain and application thereof

A collagen and collagen technology, applied in the field of genetic engineering, can solve problems such as different codon preferences, failure to meet purity requirements, insufficient protein purity, etc., to optimize secondary structure, avoid low translation efficiency, eliminate codons and develop The effect of clip structure

Active Publication Date: 2019-12-24
JIANGSU TRAUTEC MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the recombinantly expressed human collagen used for production usually does not add an affinity tag or only adds an affinity tag at one end, and the expressed mature peptide chain is easier to degrade. The protein purity obtained by traditional non-specific purification methods or single affinity tag purification Insufficient, it is difficult to remove some degradation products, and cannot meet the application with high purity requirements
In the prior art, the use of microorganisms to obtain collagen also has the following disadvantages: the DNA sequences expressing collagen are all natural coding sequences in the human genome (generally obtained by reverse transcription methods), but microorganisms and humans are involved in the process of transcription and translation of genes. There are certain differences among them, especially in the codon preference. Using the natural coding DNA sequence of human collagen to express in microorganisms will cause low translation and expression efficiency due to codon preference and mRNA structure.

Method used

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  • Recombinant humanized III type collagen alpha 1 chain and application thereof
  • Recombinant humanized III type collagen alpha 1 chain and application thereof
  • Recombinant humanized III type collagen alpha 1 chain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] The full length of the recombinant human type III collagen is 1082 amino acids, with a 6His tag at the amino end and a Strep tag at the carboxyl end. The amino acid sequence of the recombinant human type III collagen is shown in SEQ ID NO.1.

[0076] The preparation method is as follows:

[0077] 1. Construction of recombinant expression vectors (such as figure 1 shown)

[0078] 1.1 Synthesis of optimized genes

[0079] According to the gene sequence NM_000090.3 of type III collagen registered in Genebank, the gene codon was optimized according to the codon usage frequency of Pichia pastoris, and the codon with low usage rate was eliminated. At the same time, the synonymous transformation method was used to eliminate EcoRI (GAATTC), Restriction sites such as NotI (GCGGCCGC) have removed 2 GGTAAG splicing sites and 4 GGTGAT splicing sites. The optimized type III collagen gene sequence is shown in SEQ ID NO.2. The optimized type III collagen gene sequence is given by A...

Embodiment 2

[0171] Example 2 Preparation of Collagen Skin Care Products for External Use

[0172] According to the following mass ratio, dissolve each raw material with water, and stir well to form a colorless, odorless and transparent moisturizing skin care essence.

example 1

[0173] Example 1, the recombinant human type III collagen prepared in Example 1 of the present invention 0.1%; glycerol 1%; butanediol 1%; sodium hyaluronate 0.1%; carbomer 0.5%; betaine 1%; silk peptide 0.05%; β-glucan 0.2%; jojoba ester 0.1%; dipotassium glycyrrhizinate 0.3%; triethanolamine 0.1%; the rest is purified water.

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Abstract

The invention discloses a recombinant humanized III type collagen alpha 1 chain and an application thereof. The nucleotide sequence for coding the recombinant humanized III type collagen alpha 1 chainis shown as SEQID NO: 1. The recombinant humanized III type collagen alpha 1 chain sequentially comprises an amino-terminal affinity purification label, a humanized III type collagen mature peptide chain and a carboxy-terminal affinity purification label from an amino terminal. According to the recombinant humanized III type collagen alpha 1 chain disclosed by the embodiment of the invention, a specificity affinity purification label is designed at two ends of the humanized III type collagen mature peptide chain, so that purification of overall-length protein is facilitated, and the recombinant humanized III type collagen alpha 1 chain can also be used for identification of collagen. According to the recombinant humanized III type collagen alpha 1 chain disclosed by the embodiment of theinvention, the III type collagen mature peptide chain is adopted, the propeptide at two ends is removed, and the expression number of the gene without propeptide is far higher than those of the gene containing the propeptide.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, more specifically, the invention relates to a recombinant human type III collagen α1 chain and its application. Background technique [0002] Collagen is the main protein in all connective tissues including skin, bones, tendons and cartilage. For example, it forms large fiber bundles in the skin, each fiber bundle contains many individual collagen fibrils, making the skin structure strong and flexible. Collagen, a mature triple helix, is abundant in the human body and is valuable for a range of industrial and medical applications. Collagen has been widely used in clinical applications, and it has been proven safe and effective in soft tissue expansion, wound and burn repair, orthopedics and cardiovascular. [0003] Human type III collagen (hCOL3A1) is a fibril-forming collagen that is widely distributed in stretchable connective tissues such as skin, internal organs or vascular syst...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K14/78C12N15/62C12N15/81C12N1/19A61L15/32A61L15/42A61K8/65A61Q19/00C12R1/84
CPCA61K8/65A61L15/325A61L15/42A61Q19/00C07K14/78C07K2319/21C07K2319/22C12N15/815C12N2800/22Y02P20/55
Inventor 钱松王丽萍
Owner JIANGSU TRAUTEC MEDICAL TECH CO LTD
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