Hydroxylation method of recombinant human-derived collagen

A collagen and hydroxylation technology, applied in chemical instruments and methods, animal/human proteins, biochemical equipment and methods, etc., can solve the problem of high expression of collagen produced by recombinant bacteria, harsh exogenous expression conditions, and large-scale application problems such as suitable for large-scale production, easy high-throughput expression, and simple structure

Inactive Publication Date: 2018-12-18
XIAN GIANT BIOGENE TECH CO LTD
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  • Abstract
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AI Technical Summary

Problems solved by technology

Although the above methods can achieve the hydroxylation of collagen, the former requires a hyperosmotic culture environment, which is not easy to produce recombinant bacteria and high expression of collagen, and the latter requires the co-expression of human proline hydroxylase, which has 4 The subunit structure is complex, exogenous expression conditions are harsh, and the expression activity is low, which cannot be applied on a large scale

Method used

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  • Hydroxylation method of recombinant human-derived collagen
  • Hydroxylation method of recombinant human-derived collagen
  • Hydroxylation method of recombinant human-derived collagen

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preparation example Construction

[0043] According to the above method, the present invention also relates to a method for preparing hydroxylated recombinant human-derived collagen, comprising the following steps:

[0044] (1) Construct a co-expression strain of the human collagen gene and the proline hydroxylase gene derived from Cystis spp. RH1, the gene sequence is SEQ ID No.1, and the corresponding amino acid sequence is SEQ ID No.2;

[0045] (2) Express collagen and proline hydroxylase;

[0046] (3) Separation and purification of collagen to obtain hydroxylated recombinant collagen.

[0047] Step (1) is specifically:

[0048] (1.1) Obtain the collagen gene and the proline hydroxylase gene derived from the cystic fungus RH1 by means of PCR or whole gene synthesis;

[0049] (1.2) Select the appropriate expression system and construct the corresponding plasmid;

[0050] (1.3) Transfer the plasmid into the expression host;

[0051] (1.4) Screen positive transformants to obtain co-expression strains.

[0...

Embodiment 1

[0058] Example 1: Preparation of Hydroxylated Recombinant Human Type III Collagen Alpha Chain Protein

[0059] 1. Co-expression strain construction

[0060] 1) p Construction of KK223-3-TPH: the proline hydroxylase gene (TPH) derived from the cystic fungus RH1, the gene sequence is SEQ ID No.1, and the corresponding amino acid sequence is SEQ ID No.2 through whole gene synthesis After synthesis, it is directly connected to the pKK223-3 plasmid p KK223-3-TPH.

[0061] ) p Construction of BV220-COL3A1: Total RNA was extracted from fresh human placenta tissue, and a cDNA library was established by reverse transcription. Using the cDNA library as a template, the human type III collagen α chain gene CO3A1 was amplified by PCR with the following primers (GenBank: AAH28178.1 ), the PCR product was double digested and ligated into the vector p BV220 was transformed into Escherichia coli and the positive transformant was picked to extract the plasmid. p BV220-COL3A1. The PCR pri...

Embodiment 2

[0078] Example 2 Hydroxylated recombinant human Collagen type α chain protein preparation

[0079] 1. Co-expression strain construction

[0080] 1) p PICZB-TPH build: with p The KK223-3-TPH plasmid was used as a template to amplify the TPH gene and connect it with the EcoRI restriction site p Among the PICZB plasmids, positive clones were screened, and the plasmids were extracted. p PICZB-TPH plasmid.

[0081] ) p PIC9k-COL1A1 construction: total RNA was extracted from fresh human placenta tissue, a cDNA library was established by reverse transcription, and the cDNA library was used as a template to amplify human Collagen type α chain gene COL1A1 (Gene ID: 1277), the PCR product was double digested and ligated into the vector p PIC9k was transformed into Escherichia coli and the positive transformants were picked to extract the plasmid. p PIC9k-COL1A1.

[0082] ) Preparation of co-expression strains

[0083] Will p PICZB-TPH and p PIC9k-COL1A1 was linearized wit...

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Abstract

The invention relates to a hydroxylation method of a recombinant human-derived collagen; a collagen is hydroxylated by co-expression of proline hydroxylase from cystis bacteria RH1 and a human-derivedcollagen gene; the proline hydroxylase of the cystis bacteria RH1 has a gene sequence shown in SEQ ID No.1 and has a corresponding amino acid sequence shown in SEQ ID No.2. The invention provides a hydroxylation method of the recombinant collagen, wherein the method is easy to realize and simple in preparation process. The recombinant collagen prepared by the method is more similar to natural collagen in amino acid composition and spatial structure, and has the activity more similar to that of the natural collagen. Compared with a recombinant collagen prepared by a conventional method, the recombinant human-derived collagen allows the application field to be expanded, allows more excellent materials to be chosen in the increasingly developed fields of medical devices, cosmetics and food,and is more beneficial.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular relates to a hydroxylation method of recombinant human collagen. Background technique [0002] Collagen is a biopolymer protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. It is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging, etc. It is one of the most critical raw materials in the biotechnology industry. It is used in medical materials, cosmetics, food It is widely used in industry. [0003] At present, the collagen used in industrialization is mainly extracted from the skin or bones of animals through acid, alkali or enzymatic methods. The main source is animal connective tissue, but the collagen extracted from ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K14/78C12N15/70C12N15/81
CPCC07K14/78C12N9/0071C12N15/70C12N15/815C12Y114/11002
Inventor 范代娣宇文伟刚马晓轩马沛米钰
Owner XIAN GIANT BIOGENE TECH CO LTD
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