Preparation method of water-soluble human collagen VI polypeptide

A human collagen, water-soluble technology, applied in animal/human protein, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of easy infection of pathogenic microorganisms, raw materials with low extraction rate, and difficult to obtain.

Inactive Publication Date: 2011-05-18
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the production of type VI human collagen has not been reported, and the placental tissue method greatly limits the large-scale preparation of collagen due to the susceptibility of pathogenic microorganisms, low extraction rate and unavailability of raw materials.

Method used

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  • Preparation method of water-soluble human collagen VI polypeptide
  • Preparation method of water-soluble human collagen VI polypeptide
  • Preparation method of water-soluble human collagen VI polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 A kind of preparation method of water-soluble type VI human collagen polypeptide:

[0054] I. Using the plasmid pCMV-SPORT6 containing the human COLVIA2 mRNA gene sequence as a template, the water-soluble VI human collagen polypeptide target gene was cloned by PCR

[0055] (1) Preparation of plasmid DNA from Escherichia coli containing plasmid pCMV-SPORT6: follow the instructions of the plasmid extraction kit from Omega to extract and prepare;

[0056] (2) According to the full-length mRNA sequence of Human Collagen type VIA2 Accession Number: BC065509.1 in Genebank (Genebank) No. BC065509.1, design the upstream and downstream primers with restriction sites ECORI and SalI as follows:

[0057] COL6A2ECORI is 5'atcGAATTCGGCAACAAAGGAGCCAAG3'

[0058] COL6A2SalI is 5'atcGTCGACGTTCTTGACAGCCTCCTT3'

[0059] The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min...

Embodiment 2

[0106] Example 2 A method for preparing a water-soluble type VI human collagen polypeptide. In the step II (2), the water-soluble type VI human collagen target gene is connected to the plasmid pET-32a, and the E.coli DNA of TAKARA is used Ligase ligase was reacted in a water bath at 16° C. for 2 hours to obtain a recombinant vector; other steps and conditions were the same as in Example 1.

Embodiment 3

[0107] Example 3 A method for preparing a water-soluble type VI human collagen polypeptide, in the step II (2) the water-soluble type VI human collagen target gene is connected to the plasmid pET-32a, and the E.coli DNA of TAKARA is used The Ligase ligase was reacted in a water bath at 16° C. for 3 hours to obtain the recombinant vector; other steps and conditions were the same as in Example 1.

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Abstract

The invention provides a preparation method of a water-soluble human collagen VI polypeptide, which comprises the following steps: linking coding genes of the human collagen VI polypeptide into a cloning vector, transforming Escherichia coli, constructing engineering bacteria, inducing the engineering bacteria to express the human collagen VI polypeptide, and respectively establishing an engineering bacteria fermentation process and a target protein purification process. The protein electrophoretic analysis and activity verification indicate that the molecular weight of the human collagen VI polypeptide is 46kDa, and the human collagen VI polypeptide is soluble in supernatant, has favorable water solubility and exists in the form of a monomer or polymer. The human collagen VI polypeptide in vitro can protect cells from ultraviolet injury, and has the functions of diminishing inflammation and moistening. The invention realizes a large-scale gene engineering preparation method of a water-soluble human collagen VI polypeptide.

Description

technical field [0001] The invention relates to a method for preparing a water-soluble type VI human collagen polypeptide, in particular to a method for preparing a water-soluble type VI human collagen polypeptide through genetic engineering. Background technique [0002] Collagen is an extremely important protein in the structure of connective tissue. In the human body, it mainly exists in skin, bone, cartilage, teeth, muscle bonds, ligaments and blood vessels. Due to the inherent biocompatibility, biodegradability and functions of promoting cell growth of collagen, it is widely used in medicine, cosmetology, Applications in fields such as cosmetics and food continue to expand. [0003] The current main method of producing collagen is to extract it from animal connective tissue (pig skin, cowhide, donkey skin, fish skin, etc.) by acid and alkali hydrolysis, and part of the biological activity is easily lost during the extraction process, and the extracted product component...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/70C07K14/78C07K1/22
Inventor 陈光吴铭王刚孙孙旸
Owner JILIN AGRICULTURAL UNIV
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