Collagen

Abstract

The invention relates to a collagen, which has an amino acid sequence of gvkg acgldgkg, dkgagppgrpgglaghgkg, vppgqsgpgpgpgpgpgrgpgpgpgpgg, vdgg, rgg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg, gg,

Description

Technical Field

[0001] The present invention relates to the field of collagen, in particular to collagen. Background Art

[0002] Collagen is one of the most abundant proteins in the human body. It is needed in various parts of the body, including around the eyes, mouth, armpits, arms, abdomen, etc. It is responsible for maintaining the stability and strength of body tissues by forming a support network on the cell structure.

[0003] Extracting collagen from animals has disadvantages such as potential risks of viruses and allergies, as well as complicated post-processing. Collagen is prepared using recombinant DNA technology. The human collagen gene sequence is used as a template, all or part of the gene fragments are cloned and the fragments are optimized and improved. After being transferred into host cells, the protein encoded by the target gene fragment is expressed by fermentation, and recombinant humanized collagen with higher purity is obtained through purification.

[0004] However, the biosynthesis of recombinant humanized collagen is currently mainly carried out in Escherichia coli and Pichia pastoris. Although the collagen produced by the former has a high yield, there is a risk of endotoxins in the product, and the post-fermentation processing is relatively cumbersome. The use of yeast to produce collagen has the problem of using methanol to induce expression during fermentation, which poses a certain degree of production safety risk.

[0005] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention discloses collagen.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A collagen amino acid sequence is shown as SEQ ID NO 1.

[0009] Furthermore, the secretory expression yield is greater than 2.5 g / L.

[0010] The beneficial effects of the present invention are as follows:

[0011] 1. The collagen protein adopts the amino acid sequence disclosed in the present invention, and the recombinant collagen protein obtained greatly improves the yield of recombinant expression and the subsequent purification yield.

[0012] 2. Recombinant collagen contains a large amount of amino acids and hydrophilic groups, and its molecular structure is highly similar to human collagen, which makes recombinant collagen less likely to cause rejection reactions when used, reducing the risk of use.

[0013] 3. There is no need to use methanol to induce and reduce the safety hazards caused by it. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of collagen expression.

[0015] Figure 2 It is a bar graph of collagen fermentation yield. DETAILED DESCRIPTION

[0016] The specific implementation of the present invention will be described below in conjunction with the accompanying drawings.

[0017] Embodiment 1:

[0018] The amino acid sequence of a collagen is shown as SEQ ID NO 1.

[0019] The specific amino acid sequence is:

[0020] Gvkgacgldgekgdkgeagppgrpglaghkgemgepgvpgqsgapgkegligpkgdrgfdgqpgpkgdqgekgergtpgiggfpgpsgndgsagppgppgsv gppglrgdpgfegergkpglpgekgeagdpgrpgdlgpvgyqgmkgekgsrgekgsrgpkgykgekgkrgidgvdgvkgemgypglpgckgspgfdgiqgpp gppgppgpagkeglrgprgdqgpvgrtgevgavgppgfagekgpsgeagtagppgtpgpqgllgapgilglpgsrgerglpgvagavgepgplgiagppgar gssgdpgkpgeagnagvpgqrgapgkdgevgpsgpvgppglagergeqgppgptgfqglpgppgppgeggkpgdqgvpgdpgavgplgprgergnpgergep.

[0021] Embodiment 2:

[0022] The collagen prepared in Example 1 is used as one of the raw materials of biomaterials, and the biomaterials include cartilage repair tissue materials, skin repair tissue materials and blood vessel repair tissue materials.

[0023] Embodiment 3:

[0024] The collagen prepared in Example 1 is used as the raw material of the drug carrier, and the drug carrier includes a capsule shell, a patch, a spray and a gel.

[0025] Embodiment 4:

[0026] The collagen prepared in Example 1 is used as a raw material for cosmetics, and the cosmetics include lotion, facial mask, facial cream, foundation and toner.

[0027] Collagen Expression:

[0028] Platform ID: Bio-52827.

[0029] Specifications: Glycerol / culture.

[0030] Latin genus name: Corynebacterium Glutamicum.

[0031] Strain name: Corynebacterium glutamicum.

[0032] Other numbers: ACCC16522=ATCC13032.

[0033] Medium number: 2.

[0034] Cultivation temperature: 30℃.

[0035] Cultivation time: 24-48 hours.

[0036] Secretory expression results,

[0037] like Figure 1 As shown, band 1 is a protein molecular weight marker, and band 2 is collagen sequence 1 (fermented for 30 hours, the fermentation broth supernatant was diluted four times and loaded).

[0038] Calculated by liquid chromatography:

[0039] The secretory expression yield of sequence 1 reached 2.7 g / L.

[0040] Proven that collagen increases the production of cell expression.

[0041] Expression yield experiment:

[0042] 1. Prepare basal culture medium:

[0043] Weigh 6 g of tryptone, 3 g of yeast extract powder, 6 g of sodium chloride, and 6 g of brain heart extract powder, dissolve them in purified water to make up to 600 mL, divide them into 2 1 L shaking flasks, 300 mL in each bottle, and sterilize them at 116±1℃ for 16 min for later use.

[0044] 2. Fermentation:

[0045] The positive clones of Corynebacterium glutamicum containing the target sequence were activated on plates, single clones were picked, and shake-flask cultured at 30 degrees. They were then inoculated into 3-5L fermenters and fermented for about 30 hours according to conventional fermentation processes.

[0046] 3. Canning treatment:

[0047] The fermentation broth was centrifuged at 4°C for 30 min to remove the bacteria. The supernatant was filtered through a 0.22 um filter membrane and then subjected to liquid phase detection. The fermentation yield was calculated based on the standard curve.

[0048] 4. Result statistics:

[0049] 1) Control: Select the recombinant collagen products currently available on the market (sequences have been made public), construct a recombinant expression-positive strain of Corynebacterium glutamicum according to the same molecular construction strategy, and use the same activation, fermentation, and detection methods to compare yields.

[0050] 2) Test yield results: Figure 2 shown.

[0051] The unit yield of the control group was about 1 g / L, and the unit yield of the collagen products of the three experimental groups was above 2.5 g / L, which proved that the recombinant collagen disclosed in the present invention greatly increased the yield of recombinant expression.

[0052] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0053] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A collagen protein, characterized in that The amino acid sequence of the collagen is shown as SEQ ID NO 1.

2. The collagen according to claim 1, characterized in that: The secretory expression yield is greater than 2.5g / L.

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