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Recombinant humanized collagen and its industrial production method and product application

A collagen and humanization technology, applied in the fields of protein purification and genetic engineering, can solve the problems of water-solubility and hydrophilicity, moisturizing and hygroscopicity, etc., and achieve a significant effect of promoting proliferation, simple control and good effect. Effect

Active Publication Date: 2022-05-27
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 and its industrial production method and product application
  • Recombinant humanized collagen and its industrial production method and product application
  • Recombinant humanized collagen and its industrial production method and product application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of rHLC Escherichia coli strain

[0048] 1. Obtaining rHLC genes with different repeat numbers

[0049] The rHLCs with different numbers of repeat units were reverse-translated for the amino acid sequence and optimized for E. coli. GenScript Biotechnology Co., Ltd. was entrusted to synthesize their corresponding DNA sequences, and NcoI and 3 were introduced at the 5' end of the DNA during synthesis. ' end introduced XhoI restriction site. The synthesized genes were individually 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 methods in "Molecular Cloning", and then the recombinant expression vector was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock transformation.

[0052] 3. Expres...

Embodiment 2

[0054] Example 2 rHLC small-scale fermentation (medium volume 10L)

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

[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 the LB solid medium containing 50 μg / ml kanamycin using ...

Embodiment 3

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

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

[0066] 1. Obtain secondary seed solution in the same way as in Example 2.

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

[0068] 3. Prepare 150L basal culture based on in-situ sterilization in a 5...

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Abstract

The invention relates to recombinant humanized collagen and its preparation method and application. The amino acid sequence of the humanized collagen (rHLC) of the present invention is shown in SEQ ID NO: 1, and this sequence is used as a core unit to repeat 3-25 times in series to form a series of humanized collagen molecules with different molecular weights. The rHLC molecular sequence of the present invention is derived from human type I collagen, has great water solubility and hydrophilicity, and has good moisture retention, hygroscopicity and biocompatibility, and has a significant effect of promoting proliferation; in Escherichia coli host High-efficiency expression; the fermentation process medium cost is low, the composition is clear, the fermentation cycle is short, the control is simple, and the process is easy to scale up; the purification method is simple to operate, low in cost, good in effect, stable in process, and the purified product has purity, endotoxin, HCP, DNA residues, etc. can meet the pharmaceutical standards, and the production process can meet the needs of industrial production, and can be applied to medical 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 method for expression and purification of recombinant humanized collagen molecules (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. Supports organs and protects the function of the body. There are many types of collagen, the common types include: type I, type II, type III, type V and type XI, among which type I collagen mainly exists in the skin, tendons and other tissues, and is the most abundant type of collagen. Collagen has good biocompatibility, biodegradability and biological activity (promoting wound recovery, coagulat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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