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Recombinant human collagen and application thereof

A technology of human collagen and collagen, which is applied in the field of genetic engineering, can solve the problems of unsuitable long-fragment peptide chain full-length detection, complicated purification process operation, and difficulty in obtaining collagen products, so as to avoid the limitation of utilization efficiency, Elimination of codons and hairpin structures, high biocompatibility and biosafety

Inactive Publication Date: 2020-04-14
JIANGSU TRAUTEC MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, molecular sieves and ion exchange resins are mostly used for separation and purification of collagen, but collagen is difficult to separate due to its unique G-X-Y structure, especially with the degradation fragments of the same collagen, and the purification process is complicated and difficult to operate. The purification process between different batches is difficult to stabilize, resulting in the collagen short peptide products on the market are mostly a mixture of short peptides and their degradation products, it is difficult to obtain collagen products with a single peptide chain, which limits its wide application
[0008] At present, the detection of collagen in products mainly uses biological mass spectrometry to study the type and content of collagen. This method can only detect whether it is collagen, but cannot confirm whether it is a full-length fragment, so it is not suitable for the full-length of long fragment peptide chains. detection

Method used

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  • Recombinant human collagen and application thereof
  • Recombinant human collagen and application thereof
  • Recombinant human collagen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The full length of the recombinant human collagen is 1056 amino acids, with a Flag tag at the N-terminal and a 6His tag at the C-terminal. The amino acid sequence of the recombinant human collagen is shown in SEQ ID NO.1.

[0065] The preparation method is as follows:

[0066] 1. Construction of recombinant plasmids

[0067] 1.1 Synthesis of optimized genes

[0068] According to the amino acid sequence of human type I collagen α2 chain mature peptide and its corresponding gene sequence registered in Genbank, referring to the preferred codons of Pichia pastoris, the DNASTAR software was used to optimize the gene without changing the original amino acid sequence of collagen ColIα2 Sequence, and removed the restriction endonuclease sites such as ApaI, Bgl II, BamH I, EcoR I, Not I, Sac I, Sal I, the obtained gene sequence is shown in SEQ ID NO.2, and then provided by Nanjing GenScript Synthetic Technology Co., Ltd.

[0069] 1.2 Construction of recombinant plasmids by PC...

Embodiment 2

[0118] Example 2 Identification of Recombinant Human Collagen

example 1

[0120] 1. Western blotting detection

[0121] 1) Take the fermentation supernatant and carry out SDS-PAGE electrophoresis until the bromophenol blue completely runs out from the lower edge of the separation gel;

[0122] 2) Take out the gel from the glass plate and soak it in the transfer buffer;

[0123] 3) Take a PVDF membrane with the same size as the gel, soak it in anhydrous methanol for 10 seconds, transfer it to the membrane transfer buffer and continue soaking for later use;

[0124] 4) Place the sponge, filter paper, PVDF membrane, gel, filter paper, and sponge pre-soaked in the transfer buffer in the transfer folder in order to ensure that there are no air bubbles between each layer;

[0125] 5) Put the transfer clip into the electroporation tank, the membrane is close to the positive electrode, the glue is close to the negative pole, and the electroporation tank is placed in an ice water bath, 450mA transfer for 2h;

[0126] 6) Take out the PVDF membrane, soak it ...

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Abstract

The invention provides recombinant human collagen and an application thereof. An amino acid sequence for encoding the recombinant human collagen is shown as SEQ ID NO: 1, and the recombinant human collagen sequentially comprises an amino terminal affinity purification tag, a human collagen type I alpha2 mature peptide chain and a carboxyl terminal affinity purification tag. According to the recombinant human collagen provided by the embodiment of the invention, the specific affinity purification tags are designed at the two ends of the human collagen type I alpha2 mature peptide chain, so thatthe recombinant human collagen is easy to separate, purify and detect, has higher molecular weight and special physicochemical properties, and is easy to ferment and express, high in yield and wide in application.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, more specifically, the invention relates to a recombinant human collagen and its application. Background technique [0002] The aging of the human body is manifested as changes in skin conditions, such as sagging and wrinkling. The condition of the skin directly affects the aesthetic appearance and mental state. The content of collagen in the skin is as high as 75%, which maintains the elasticity and moisture of the skin. Collagen loss is the main cause of skin aging. Improve the collagen content in the skin, help the skin resist aging and restore skin elasticity. [0003] Type I collagen is the collagen with the most extensive tissue distribution and the highest content among the 29 collagens that have been discovered so far, accounting for about 90% and about 27% of the total protein in the animal body. In the morphogenesis and cell metabolism of new tissues, it endows new tissu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C12N15/81A61K8/65A61Q19/00A61L15/32A61L15/42A61L26/00
CPCA61K8/65A61L15/325A61L15/42A61L26/0033A61L26/008A61Q19/00C07K14/78C12N15/815
Inventor 钱松张永正
Owner JIANGSU TRAUTEC MEDICAL TECH CO LTD
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