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Recombinant humanized collagen as well as industrial preparation method and product application thereof

A collagen and humanized technology, applied in the fields of genetic engineering and protein purification, can solve the problems of water-solubility and hydrophilicity, moisturizing and hygroscopicity, etc.

Active Publication Date: 2021-05-04
GENSUN INST OF BIOMEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are already some recombinant collagen products on the market, and the existing products are all designed according to the characteristics of their respective products, based on the human collagen sequence, but these designs cannot meet the requirements of great water solubility and affinity. Special performance requirements such as water-based, good moisture retention and hygroscopicity, and also need to meet the needs of industrial production

Method used

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  • Recombinant humanized collagen as well as industrial preparation method and product application thereof
  • Recombinant humanized collagen as well as industrial preparation method and product application thereof
  • Recombinant humanized collagen as well as industrial preparation method and product application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The construction of embodiment 1rHLC escherichia coli bacterial strain

[0048] 1. Acquisition of rHLC genes with different repeat numbers

[0049] rHLC with different numbers of repeating units reverse-translated the amino acid sequence and optimized it for Escherichia coli, entrusted GenScript Biotechnology Co., Ltd. to synthesize the corresponding DNA sequence, and introduced NcoI and 3 at the 5' end of the DNA during synthesis. ' end introduced XhoI restriction site. The synthesized genes were respectively cloned into the pUC57-T-simple vector and delivered as recombinant plasmids.

[0050] 2. Construction of recombinant expression strains

[0051] The rHLC gene was cloned into the pET28a vector to form a recombinant expression vector using the double enzyme digestion and enzyme ligation method in "Molecular Cloning", and then the recombinant expression vector was transformed into Escherichia coli BL21 (DE3) competent cells using the heat shock transformation meth...

Embodiment 2

[0054] Embodiment 2 rHLC small test fermentation (medium volume 10L)

[0055] This example mainly describes the 10L fermentation process of rHLC. In the fermentation process, glycerol with definite composition is used as the carbon source for cell metabolism, and ammonia water is used (both for pH adjustment and nitrogen source for cell metabolism in the fermentation process) and diammonium hydrogen phosphate as the nitrogen source for bacterial metabolism; at the same time, in order to promote bacterial growth, shorten the fermentation cycle, and increase fermentation yield, compound nutrients such as yeast powder and peptone are added to the medium. The fermenter used was a Sartouris cplus fermenter, and the initial volume of fermentation was 10 L.

[0056] Specific steps are as follows:

[0057] 1. Preparation of fermented seed liquid: inoculate the rHLC Escherichia coli strain cryopreservation tube constructed in Example 1 into LB solid medium containing 50 μg / ml kanamyci...

Embodiment 3

[0064] Example 3 rHLC pilot scale-up fermentation (150L)

[0065] This example describes the scale-up of the fermentation process of rHLC, on the basis of the small-scale fermentation process (Example 2), the research and verification of the pilot-scale scale-up fermentation process is carried out. details as follows:

[0066] 1. Obtain the secondary seed liquid with the method of embodiment 2.

[0067] 2. Prepare 15L of basic culture according to the method in Example 2 and the batch medium components to sterilize in situ based on a 50L seed tank, then inoculate the secondary seed liquid in step 1 into the seed tank for cultivation, and the culture conditions are: : Tank pressure = 0.3MPa T = 37°C D0 = 40% pH = 7.0, use ammonia water to control the pH not lower than 7.0, and do not add antibiotics during the cultivation process. It can be transplanted to working tanks after being cultivated to OD=15-30. As a tertiary seed solution.

[0068] 3. Prepare 150L of basic cultur...

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Abstract

The invention relates to recombinant humanized collagen as well as an industrial preparation method and product application thereof. The invention provides humanized collagen (rHLC) derived from human type I collagen. An amino acid sequence of the humanized collagen (rHLC) is as shown in SEQ ID NO: 1, and the sequence can be used as a core unit to perform series connection of 3-25 repetitions to form a series of humanized collagen molecules with different molecular weights. An rHLC molecular sequence designed by the humanized collagen is from the human type I collagen, has great water solubility and hydrophilicity, meanwhile, has good moisture retention, hygroscopicity and biocompatibility, and has a remarkable proliferation promoting effect; designed rHLC molecules can be efficiently expressed in an escherichia coli host, and the yield reaches 0.5-1.5 g / L; an rHLC fermentation process is low in medium cost, clear in component, short in fermentation period, simple to control and easy to scale up; and a developed rHLC purification method is simple to operate, low in cost, good in effect and stable in process, the purity, endotoxin, HCP, DNA residues and the like of a pure product after purification can meet the medicinal standards, the production process can meet the industrial production requirements, and the humanized collagen can be applied to medical beauty cosmetics or functional cosmetics.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and protein purification, and in particular relates to a preparation method for expression and purification of recombinant human like collagen (recombinant human like collagen, rHLC for short). Background technique [0002] Collagen is the most abundant protein in the human body, accounting for 25-30% of the total protein in the human body. It mainly exists in the skin, bones, cartilage, teeth, tendons, and ligaments of the human body. It is an extremely important structural protein in connective tissue. Support the organs and protect the functions of the body. There are many types of collagen, and the common types include: type I, type II, type III, type V, and type XI. Among them, type I collagen mainly exists in skin, tendon and other tissues, and is the most abundant type of collagen. Collagen has good biocompatibility, biodegradability and bioactivity (promoting wound recovery, blood coag...

Claims

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Application Information

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IPC IPC(8): C07K14/78C12N15/70C12N1/21C07K1/36C07K1/30C07K1/34A61K8/65A61Q19/00C12R1/19
CPCC07K14/78C12N15/70A61K8/65A61Q19/00
Inventor 马永赵百学王安良
Owner GENSUN INST OF BIOMEDICINE
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