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Recombinant human collagen containing hydroxyproline and hydroxylysine and production method of recombinant human collagen

A technology of human-like collagen and hydroxylysine, applied in the direction of animal/human protein, biochemical equipment and methods, peptide/protein components, etc. To achieve the effect of promoting cell proliferation and promoting adhesion, accelerating wound hemostasis and healing, and promoting the repair of epidermal cells

Active Publication Date: 2020-06-26
肽源(广州)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

[0003] Today, the main source of collagen is still animal-derived collagen, such as seafood such as fish and livestock processing by-products such as cattle and pigs, which are obtained through extraction processes such as acid, alkali, and enzymes. However, animal-derived collagen has the following obvious deficiencies: 1) The composition of the final product is complex, the structure of the active ingredient is not clear, and the active structure is easily destroyed during the intense extraction process; 2) The non-human protein is immunogenic, and the risk of allergic reaction cannot be ignored; 3) There are prions such as mad cow disease Virus risk, and the issue of religious belief is difficult to solve
Although this method solves the problem of partial hydroxylation of recombinant collagen to a certain extent, it also has the following shortcomings: 1) The structure and function design of recombinant human collagen is still unclear; 2) For Escherichia coli with complete hydroxylation , Escherichia coli is a non-GRAS expression host (Generally Recognized as Safe, which is an indicator for the US FDA to evaluate the safety of food additives). In addition, the downstream purification process is complicated and it is difficult to remove endotoxin
3) For other expression hosts, hydroxylation is limited to proline hydroxylation, and it is difficult to achieve simultaneous hydroxylation of proline and lysine

Method used

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  • Recombinant human collagen containing hydroxyproline and hydroxylysine and production method of recombinant human collagen
  • Recombinant human collagen containing hydroxyproline and hydroxylysine and production method of recombinant human collagen
  • Recombinant human collagen containing hydroxyproline and hydroxylysine and production method of recombinant human collagen

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1: Recombinant Human Collagen Nucleotide and Amino Acid Sequence Design, Construction of Expression Vector

[0041] Based on the codon preference of Kluyveromyces, the recombinant human-like collagen (hlCOL), α subunit of proline hydroxylase (P4H), and Protein disulfide isomerase (PDI), lysine hydroxylase L230 (the code name of lysine hydroxylase L230 is LH) protein gene hlCOL, P4HA, PDI, LH. The gene sequence was synthesized by Shanghai Jierui Bioengineering Co., Ltd. The nucleotide sequence of the synthetic recombinant human-like collagen (hlCOL) is shown in SEQ ID No.1, the α subunit of proline hydroxylase (P4H) The nucleotide sequence is shown in SEQ ID No.2, the nucleotide sequence of lysine hydroxylase is shown in SEQ ID No.3, and the nucleotide sequence of lysine hydroxylase is shown in SEQ ID No.4. Obtained from the nucleotide sequence encoding expression of recombinant human-like collagen (hlCOL), α subunit of proline hydroxylase (P4H), protease disulfid...

Embodiment 2

[0052] Example 2: Construction of hydroxylated collagen-like engineering bacteria

[0053] Such as figure 1 , 2 , 3, and 4, use restriction endonuclease SacII (U.S. NEB Company) to linearize plasmid A, plasmid B, plasmid C, and plasmid D, and construct an enzyme digestion reaction system as shown in Table 4:

[0054] Table 4 enzyme digestion reaction system

[0055]

[0056] The reaction conditions are 37°C, 30min. After the enzyme digestion reaction, use the gel recovery kit (Beijing Tiangen Biology) to recover the carrier fragment containing the target gene by tapping the rubber, and remove the resistance gene fragment on the carrier.

[0057] The recovered high-purity plasmid fragments were added to Kluyveromyces competent cells, incubated on ice for 2 minutes, and then electroporated (1.5kv-2.5kv). Then spread it on the YCB medium containing acetamide (5mM), culture it at 28°C for 2 days, and wait until a single colony grows on the plate, and obtain the preservation...

Embodiment 3

[0071] Embodiment 3: Hydroxyproline and hydroxylysine content determination

[0072] Hydroxyproline was carried out according to the spectrophotometer method in the Ministry of Agriculture industry standard "Determination of L-Hydroxyproline in Raw Milk" (NY / T 3130-2017).

[0073] Hydroxylysine was detected using a hydroxylysine ELISA detection kit (purchased from Shanghai Yiyan Biotechnology Co., Ltd.). The steps are as follows: Take 10 μL each of the diluted standard substance (included with the kit) or the fermentation supernatant (from Example 2), and add 40 μL of the diluent; do not add to the blank well. Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well except the blank well, seal the reaction well with a sealing film, and incubate for 60 minutes in a 37°C water bath or incubator . Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let it stand for 1 min, shake off the washing solution, pat dry on abso...

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Abstract

The invention discloses a recombinant human collagen containing hydroxyproline and hydroxylysine and a production method of the recombinant human collagen. The nucleotide sequence of the recombinant human collagen containing the hydroxyproline and the hydroxylysine is shown as SEQ ID NO.1; and the amino acid sequence of the recombinant human collagen containing the hydroxyproline and the hydroxylysine is shown as SEQ ID NO.5. The recombinant human collagen is obtained by performing co-expression on four different vectors pKLAC1-hlCOL, pKLC-P4HA, pKLC-PDI and pKLC-LH respectively by using Kluyveromyces as a base cell through a series of fermentation and purification manners. The hydroxylation rates of proline and lysine of the recombinant human collagen are similar to those of natural humantype I collagen, and reaches 30.7% and 43.1%, respectively; and compared with unhydroxylated recombinant human collagen, the recombinant human collagen provided by the invention has better functionsof promoting cell adhesion and wound healing.

Description

technical field [0001] The invention relates to the technical field of biological recombinant proteins, in particular to a recombinant humanoid collagen containing hydroxyproline and hydroxylysine and a production method thereof. Background technique [0002] Collagen is the main structural protein of vertebrates, accounting for about 30% of the total protein of organisms. More than half of the extracellular matrix proteins in human tendons, bones, and skin are composed of collagen. With its special structure and function, collagen, as an important biomaterial, has been widely used in drug delivery, surgical suture, tissue filling, nerve repair, skin beauty and other fields. [0003] Today, the main source of collagen is still animal-derived collagen, such as seafood such as fish and livestock processing by-products such as cattle and pigs, which are obtained through extraction processes such as acid, alkali, and enzymes. However, animal-derived collagen has the following o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/81C07K14/78A61K8/65A61P17/02A61Q19/00
CPCC07K14/78C12N15/815A61K8/65A61P17/02A61Q19/00A61K38/00
Inventor 黄亚东张齐肖巧学
Owner 肽源(广州)生物科技有限公司
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