CN111004319A

CN111004319A - Recombinant human collagen and application thereof

Info

Publication number: CN111004319A
Application number: CN201911093377.5A
Authority: CN
Inventors: 钱松; 张永正
Original assignee: Jiangsu Yuezhi Biology Medicine Co ltd
Current assignee: Jiangsu Yuezhi Biology Medicine Co ltd
Priority date: 2019-03-19
Filing date: 2019-11-11
Publication date: 2020-04-14
Also published as: CN110003324A

Abstract

The invention provides a recombinant human collagen and application thereof, wherein an amino acid sequence for coding the recombinant human collagen is shown as SEQ ID NO 1, and the recombinant human collagen sequentially comprises an amino terminal affinity purification label, a human I type collagen α chain mature peptide chain and a carboxyl terminal affinity purification label from an amino terminal.

Description

Recombinant human collagen and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to recombinant human collagen and application thereof.

Background

Aging of the human body is manifested by changes in the skin condition, such as sagging, wrinkling, etc. The condition of the skin directly affects the aesthetic and mental state of the appearance. The collagen content of the skin is up to 75%, and it maintains the elasticity and moisture of the skin. Loss of collagen is a major cause of skin aging. Increasing collagen content in skin, resisting aging, and restoring skin elasticity.

Type I collagen is the collagen with the most widespread tissue distribution and the highest content among 29 collagen proteins found so far, and accounts for about 90% and about 27% of the total protein of animal bodies. It gives mechanical strength and biochemical characteristics to new tissues in morphogenesis and cell metabolism of the new tissues, and plays an important role in maintaining normal physiological functions of cells, tissues and the like and repairing damage.

The type I collagen has good biocompatibility, biodegradability, no cytotoxicity, oxidation resistance and other functions, can promote cell adhesion, cell proliferation and other effects, and can be widely applied to the fields of medicine, beauty treatment, cosmetics, health care products and the like. It also has been commonly recognized in the fields of skin injury repair, cosmetic and plastic surgery, and immunity enhancement.

At present, related researches on recombinant collagen in China are many, such as northwest university and south China university, and related research reports exist, but the main research on the recombinant collagen is a collagen short peptide fragment, and no report on recombinant human type I collagen α 2 chain full-length peptide exists.

Although the peptide fragment of the recombinant type I collagen which has been circulated in the market at present has better efficacy, the function is limited due to smaller fragment and low molecular weight. The collagen type I full-length peptide has more fibronectin FN binding sites and can more easily exert the collagen functional activity; the modified polyvinyl chloride film has higher molecular weight and unique physiological and biochemical characteristics, such as easy formation of a breathable film, long degradation resistance time, higher crosslinking degree after crosslinking and more excellent performance; and the collagen short peptide can be more durably supplemented by the slow degradation of the collagen short peptide along with the time, and even can play a role of slow release when being combined with certain medicines. Therefore, in order to fully exploit the potential value, it is necessary to solve the problems of production and detection of full-length peptides.

At present, molecular sieves and ion exchange resins are mostly adopted for separating collagen, and the collagen is difficult to separate due to the unique G-X-Y structure, particularly with degradation fragments of the same collagen, the purification process is complex to operate, and the purification process is difficult to stabilize among different batches, so that collagen short peptide products in the market are mostly mixtures of short peptides and degradation products thereof, the collagen products with single peptide chains are difficult to obtain, and the wide application of the collagen is limited.

At present, collagen detection in products mainly utilizes a biological mass spectrometry technology to research the type and content of collagen, and the method can only detect collagen but cannot confirm whether the collagen is a full-length fragment, so that the method is not suitable for full-length detection of a long fragment peptide chain.

Disclosure of Invention

The present invention is directed to solving at least one of the above problems.

Therefore, the invention provides the recombinant human collagen which is convenient to purify and identify and easy to detect.

The invention also provides an application of the recombinant human collagen.

The amino acid sequence of the recombinant human collagen is shown in SEQ ID NO 1, and the recombinant human collagen sequentially comprises an amino-terminal affinity purification tag, a human type I collagen α 2 chain mature peptide chain and a carboxyl-terminal affinity purification tag from the amino terminal.

The recombinant human collagen according to the above embodiment of the present invention may further have the following additional technical features:

according to one embodiment of the invention, the amino-terminal affinity purification tag is a Flag tag and the carboxy-terminal affinity purification tag is a 6His tag.

According to one embodiment of the invention, the total length of the recombinant human collagen is 1056 amino acids.

According to one embodiment of the invention, the gene sequence of the recombinant human collagen is shown as SEQ ID NO: shown at 9.

According to one embodiment of the invention, the gene sequence of the mature peptide chain of human type I collagen α 2 chain is obtained by adjusting the codon of the native collagen gene sequence according to the usage frequency of host codon.

According to one embodiment of the invention, the host is pichia pastoris and the modification is to replace the yeast unusual codon in the native collagen gene sequence with its customary codon.

According to one embodiment of the invention, when the primer amplification construction is carried out on the recombinant human collagen, a gene sequence for coding a Flag tag sequence is introduced into the 5 'end of a collagen gene sequence after codon optimization, and a gene sequence for coding a 6His sequence is introduced into the 3' end for amplification.

According to one embodiment of the invention, the recombinant human collagen is extracted and purified from the fermentation liquor of the pichia pastoris by a Ni-NTA His bond resin and Anti-Flag affinity purification gel adsorption method.

According to one embodiment of the invention, the recombinant human collagen is applied to an external skin care product, and the external skin care product comprises, by mass, 0.01-0.1% of the recombinant human collagen according to the embodiment, 1-3% of a humectant, 1-3% of butanediol, 0.01-0.1% of sodium hyaluronate, 0.01-0.1% of xanthan gum, 0.01-0.2% of allantoin, 0.1-1% of panthenol, 0.1-0.5% of dipotassium glycyrrhizinate, 20.01-0.1% of EDTA & Na20, 0.1-0.5% of β -dextran, 0.1-0.5% of lubrabar oil, 0.1-0.3% of sodium lactate, 0.1-1% of water-soluble azone, 0.01-0.1% of silk peptide, and the balance of purified water.

According to one embodiment of the invention, the recombinant human collagen is applied to an introduced skin care product, and the mass fraction of each component of the introduced skin care product is as follows: 0.1% -0.5% of the recombinant human collagen according to the embodiment; 1% -10% of humectant; 0.1 to 0.5 percent of sodium hyaluronate; 0.01-0.05% of micromolecular sodium hyaluronate; 0.9 percent of sodium chloride; the balance being purified water.

According to one embodiment of the invention, the recombinant human collagen is applied to the preparation of the collagen gel dressing, and the mass fractions of the components of the collagen gel dressing are respectively as follows: the recombinant human collagen of the embodiment is 0.1 to 0.5 percent; 1% -10% of humectant; 0.1% -4% of carbomer; 0.1 to 10 percent of triethanolamine; the balance being purified water.

According to one embodiment of the invention, the method is used for preparing the original protein plaster dressing, and the mass fractions of the components of the original protein plaster dressing are respectively as follows: the recombinant human collagen of the embodiment is 0.1 to 0.5 percent; 1% -10% of humectant; 0.1% -2% of xanthan gum; the balance being purified water.

According to one embodiment of the invention, the recombinant human collagen is applied to preparation of the collagen-based skin mucosa protective agent dressing, and the mass fractions of the components of the collagen-based skin mucosa protective agent dressing are respectively as follows: 0.1% -1% of the recombinant human collagen according to the embodiment; 0.1 to 1 percent of sodium hyaluronate; 0-40% of a humectant; the balance being purified water.

The recombinant human collagen provided by the embodiment of the invention has at least the following technical effects:

(1) the recombinant human collagen of the invention carries dual specificity labels at two ends, not only can obtain full-length collagen through affinity purification, but also can identify the state and content of the recombinant human collagen in a product through sandwich ELISA test;

(2) the gene sequence of the recombinant human collagen is subjected to codon optimization according to the conventional codon of pichia pastoris, so that an uncommon codon and a hairpin structure are eliminated, the secondary structure of mRNA is optimized through synonymous conversion, the low translation efficiency caused by the limitation of codon utilization efficiency is avoided, and the recombinant human collagen is more suitable for expression in pichia pastoris;

(3) compared with the conventional recombinant collagen segment and gelatin, the recombinant human collagen is full-length collagen, so that the recombinant human collagen has high molecular weight, has better chemical structure and performance as a biological macromolecule, and can be used as a biomedical material;

(4) compared with the traditional collagen production method, the recombinant human collagen is humanized recombinant collagen expressed by a eukaryotic expression system, can be secreted outside cells, has no virus and endotoxin hidden danger, avoids immunological rejection, and has higher biocompatibility and biological safety, more environment-friendly process and safer product.

(5) The recombinant human collagen has good hydrophilicity and stability, the amino acid composition of the recombinant human collagen is 100 percent same as that of the corresponding part of the amino acid sequence of the natural collagen, and the structure and the biological activity of the recombinant human collagen are closer to those of the natural collagen.

(6) The recombinant human collagen provided by the invention carries the Flag tag at the N end and the 6His tag at the C end, and the bispecific affinity purification tag is utilized, so that the purification steps are simple, the product purity is high, and the use of products such as minimally invasive, plastic and cosmetic, biomedical materials and the like can be met.

Drawings

FIG. 1 is a map of a recombinant plasmid pPIC9k-colI α 2 for secretory expression of recombinant human collagen constructed according to an embodiment of the present invention;

FIG. 2 is a PCR identification electrophoresis chart of recombinant plasmid used in the preparation process of recombinant human collagen according to the embodiment of the present invention;

FIG. 3 is an SDS-PAGE analysis diagram of recombinant Pichia pastoris engineering bacteria expressing recombinant human collagen according to the embodiments of the invention;

FIG. 4 is an immunoblotting experiment of recombinant human collagen expressed by recombinant bacteria selected in the example of the present invention;

FIG. 5 shows the mass spectrum comparison result of recombinant human collagen expressed by recombinant bacteria screened according to the embodiment of the present invention;

FIG. 6 is a schematic diagram showing the results of the overall length test of recombinant human collagen expressed by the screened recombinant bacteria by sandwich ELISA (in which the degradation product is purified degradation protein containing a C-terminal label, and the target protein is purified target protein) according to the embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

The recombinant human collagen according to the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

The amino acid sequence for coding the recombinant human collagen is shown as SEQ ID NO: 1, and the recombinant human collagen sequentially comprises an amino terminal affinity purification label, a human type I collagen α 2 chain mature peptide chain and a carboxyl terminal affinity purification label from the amino terminal.

In other words, the recombinant human collagen according to the embodiment of the invention is based on the mature peptide chain of the human type I collagen α 2 chain, and the amino acid and the carboxyl terminal of the recombinant human collagen are respectively provided with affinity purification tags, so that the recombinant human collagen has a full-length peptide chain, and the chemical structure and the performance of the recombinant human collagen are superior to those of the animal type I collagen, the prokaryotic microorganism source recombinant collagen and the commercial recombinant collagen fragments and gelatin by respectively arranging the specific affinity purification double tags at the two ends of the mature peptide chain of the human type I collagen α 2 chain, and in addition, the hydrophilic amino acid content of the mature peptide of the type I α 2 chain is higher than that of the α 1 chain, so that the recombinant human collagen has potential better hydrophilicity.

According to some specific embodiments of the invention, the amino-terminal affinity purification tag is a Flag tag, and the carboxyl-terminal affinity purification tag is a 6His tag, and by adopting two-terminal parent and tag purification, a single full-length degradation-free human type I collagen α chain mature peptide chain can be obtained, and the method can be applied to the fields such as the medical field and the like which have requirements on the uniformity of products.

In an embodiment of the present invention, the total length of the recombinant human collagen is 1056 amino acids, that is, the total length of the recombinant human collagen is 1056 amino acids, and the recombinant human collagen is composed of a Flag sequence at the N-terminal, a mature peptide sequence of human type I collagen α 2 chain, and a 6His sequence at the C-terminal, and has a full-length peptide chain and better performance.

The following describes the recombinant engineering bacteria expressing the above genes and the protein preparation method.

In one embodiment of the present invention, the gene sequence of the recombinant human collagen is obtained by adjusting codons according to the usage frequency of host codons with reference to the native collagen gene sequence. Further, the host is pichia pastoris, the regulation is to replace unusual codons of yeast in the natural collagen gene sequence with the common codons, optionally, when the recombinant human collagen gene is constructed by primer amplification, a gene sequence for coding a Flag marker sequence is introduced at the 5 'end of the collagen gene sequence after codon optimization, and a gene sequence for coding a 6His sequence is introduced at the 3' end of the recombinant human collagen gene for amplification.

Preferably, the recombinant human collagen is extracted and purified from the fermentation liquor of the pichia pastoris by a Ni-NTA His Bind resin and Anti-Flag affinity purification gel adsorption method.

That is to say, the gene sequence of the recombinant human collagen is adjusted, i.e., the codons aga, cgt, ggg, ggc, gca and the like which are not commonly used by pichia pastoris in the gene sequence corresponding to the mature peptide of the human type I collagen α 2 chain are optimized to the preferred codons aga, ggt, gct and the like, and the gene sequence obtained by analyzing by using DNASTAR software is shown as SEQ ID No. 2.

Further, when the recombinant human collagen is constructed by primer amplification, a gene sequence for coding a Flag tag sequence is introduced into the 5 'end of the collagen gene sequence after codon optimization, and a gene sequence for coding a 6His sequence is introduced into the 3' end of the collagen gene sequence and then amplified so as to facilitate the purification and detection of the recombinant human collagen.

Step one, constructing recombinant plasmid

Firstly, providing a gene sequence corresponding to an optimized human type I collagen α 2 chain mature peptide, optimizing the corresponding gene sequence without changing the original amino acid sequence of collagen according to the amino acid sequence and the gene sequence registered by Genbank, wherein the optimization is performed according to pichia pastoris preferential codons, codons agg, cgt, ggg, ggc, gca and the like which are not commonly used by pichia pastoris in the gene sequence (colI α 2) corresponding to the human type I collagen α 2 chain mature peptide are optimized into preferential codons aga, ggt, gct and the like, and the gene sequence obtained by DNASTAR software analysis is shown as SEQ ID NO: 2, so that the collagen is more beneficial to expression.

Next, restriction enzyme sites such as ApaI, Bgl II, BamH I, EcoR I, Not I, Sac I, Sal I, etc. in the optimized gene sequence were deleted and synthesized by artificial whole gene synthesis.

Finally, ① uses pPIC9k plasmid as template, utilizes primer P1 (see SEQ ID NO.3)/P2 (see SEQ ID NO.4) to make PCR amplification to obtain product of about 379bp, ② uses the above-mentioned synthesized gene sequence SEQ ID NO: 2 as template, utilizes primer pair P3 (see SEQ ID NO.5)/P4 (see SEQ ID NO.6) to make PCR amplification to obtain gene sequence with about 3149bp, α 0 uses P5 (see SEQ ID NO.7)/P6 (see SEQ ID NO.8) as primer, and uses itself as template to make PCR mutual amplification to obtain gene fragment containing about 65bp 6His coding sequence, α makes plasmid pPIC9k respectively make BamHI and NotI double enzyme digestion to obtain vector fragment, ⑤ uses instant seamless kit (sang 9), ③ and ④ to make mixed reaction, utilizes Sangon) to clone the gene sequence containing GAG I and NotI double enzyme digestion to obtain recombinant protein sequence containing collagen protein sequence of collagen protein, and uses collagen gene sequence as collagen gene sequence, so as recombination protein gene sequence of collagen gene sequence, so as to obtain recombination protein gene sequence of about α, collagen protein gene sequence, and recombination protein sequence of collagen protein gene sequence of about 379-469.

Step two, constructing gene recombinant pichia pastoris engineering bacteria

The prepared recombinant plasmid pPIC9k-colI α 2 is linearized with restriction endonuclease SalI and is electrically transformed into a competent cell of Pichia pastoris SMD1168(Invitrogen), and a high-copy positive recombinant is screened by an auxotroph marker and a G418 resistance marker to obtain the Pichia pastoris genetically engineered bacterium (the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC17147, the preservation date of 2019, 1 month and 10 days, the address: Beijing Korean area Beichen Xilu No.1, No.3, and the classification name: Pichia pastoris).

Step three, preparing the recombinant human collagen

Inoculating the recombinant Pichia pastoris engineering bacteria to a YPD liquid culture medium, and activating overnight at 30 ℃ and 220 rpm; transferring the recombinant pichia pastoris engineering bacteria to a BMGY culture medium until OD600 is 2.0-6.0, and culturing at 30 ℃ and 220rpm for 16-18 h; centrifuging at room temperature at 1500g for 5min, collecting thallus, suspending thallus with 10mL BMMY culture medium until OD600 is about 1.0, placing the obtained bacterial solution in 100mL sterile triangular flask, and continuously performing shaking culture at 28 deg.C and 220 rpm; adding methanol to the culture medium every 24 hours until the final concentration is 1% for induction culture; after the fermentation culture is finished, centrifugally collecting supernatant, adsorbing recombinant collagen by using Ni-NTA His Bands resin under a non-denaturing condition, washing impurities by using a washing solution, finally washing the recombinant collagen again, and collecting eluent; desalting and concentrating the eluent by an ultrafiltration system; adsorbing the recombinant collagen by the concentrated solution through Anti-Flag affinity purification gel, washing impurities by using a washing solution, washing the recombinant collagen again, and collecting an eluent; desalting and concentrating the eluent by an ultrafiltration system, and performing vacuum freeze drying on the prepared concentrated solution to obtain the recombinant human collagen.

Therefore, the invention optimizes the codon of the type I collagen gene according to the codon preference of pichia pastoris, eliminates the unusual codon and hairpin structure, optimizes the secondary structure of mRNA through synonymous conversion, avoids the low translation efficiency caused by the limitation of the codon utilization efficiency, and is more suitable for expression in pichia pastoris.

In summary, the recombinant human collagen according to the embodiment of the present invention provides a recombinant human collagen with affinity purification tags at both ends, which can be used to obtain full-length collagen, and can also be used to identify its state and content in a product through sandwich ELISA assay, and the recombinant human collagen has a high molecular weight, is a biomacromolecule, has a better chemical structure and performance, and can be used as a biomedical material.

The preparation process and application of the recombinant human collagen of the present invention are specifically described below with reference to specific examples and fig. 1 to 6.

Example one

The total length of the gene recombinant human collagen is 1056 amino acids, the N end of the gene recombinant human collagen is provided with a Flag tag, the C end of the gene recombinant human collagen is provided with a 6His tag, and the amino acid sequence of the gene recombinant human collagen is shown in SEQ ID NO. 1.

The preparation method comprises the following steps:

1. construction of recombinant plasmids

1.1 Synthesis of optimized genes

According to the amino acid sequence of the human type I collagen α 2 chain mature peptide registered by Genbank and the corresponding gene sequence thereof, referring to pichia pastoris preference codon, on the premise of Not changing the original amino acid sequence of collagen ColI α 2, DNASTAR software is used for optimizing the gene sequence, restriction enzyme sites such as ApaI, Bgl II, BamH I, EcoR I, Not I, Sac I, Sal I and the like are removed, the gene sequence is obtained and is shown in SEQ ID NO.2, and then the gene sequence is synthesized by Nanjing Kingrui biotechnology Limited company.

1.2 construction of recombinant plasmids by PCR, restriction, ligation, and the like

① PCR amplification is carried out with pPIC9k plasmid as template, primer P1 (SEQ ID NO.3)/P2 (SEQ ID NO.4) to obtain product of about 379bp, ② PCR amplification is carried out with synthesized gene sequence SEQ ID NO.2 as template with primer P3 (SEQ ID NO.5)/P4 (SEQ ID NO.6) to obtain gene sequence of about 3149bp, α PCR amplification is carried out with P5 (SEQ ID NO.7)/P6 (SEQ ID NO.8) as primer with self as template to obtain gene fragment containing 6His coding sequence of about 65bp, plasmid pPIC9 84 and NotI double digestion are carried out with plasmid to obtain vector fragment, ⑤ the fragments named as ①, ②, BamHI ③, ④ are mixed in certain proportion, sequenced through seamless cloning kit (Sangon), 50 deg.C double digestion is carried out with Noti double digestion to obtain vector fragment, PCR amplification is carried out with PCR amplification carried out with primer sequence of gene fragment containing 6His coding sequence, PCR amplification is carried out with plasmid DNA sequence, PCR amplification is carried out with PCR amplification, PCR amplification is carried out with plasmid DNA sequencing is carried out with PCR amplification reaction on plasmid DNA fragment containing 6 bp, PCR amplification plasmid DNA fragment containing cDNA sequence of SEQ ID NO.1, PCR amplification plasmid DNA fragment containing cDNA of about 65bp, PCR amplification plasmid DNA fragment containing cDNA of SEQ ID NO.2, PCR amplification plasmid DNA of SEQ ID NO.2, PCR amplification plasmid DNA of SEQ ID NO.1, PCR amplification plasmid of Escherichia colI strain, PCR amplification plasmid DNA of Escherichia colI strain.

2. Construction of gene recombinant Pichia pastoris engineering bacteria

2.1 linearization of the recombinant expression plasmid pPIC9k-colI α 2

Extracting recombinant plasmid pPIC9k-colI α 2, carrying out enzyme digestion at 37 ℃ by using restriction enzyme SalI, detecting whether the recombinant plasmid is completely cut by using 1% agarose gel electrophoresis, treating the enzyme digestion solution by using a gel recovery kit after the recombinant plasmid is completely cut, recovering the linearized plasmid, and desalting.

2.2 preparation of Pichia pastoris SMD1168 competent cells

1) Picking a single colony of yeast SMD1168, inoculating the single colony into a triangular flask containing 5mL YPD liquid culture medium, and carrying out shaking culture at 30 ℃ and 250rpm for overnight;

2) inoculating 50uL of the overnight culture into a 300mL triangular flask containing 50mL of fresh YPD liquid culture medium, and carrying out shaking culture at 30 ℃ and 250rpm overnight until the OD600 value reaches 1.1-1.3;

3) centrifuging the culture at 4 deg.C and 1500 Xg for 5min, and resuspending the thallus with 50ml of ice-pre-cooled sterile double distilled water;

4) centrifuging according to the step 3), and resuspending the thalli by using 25ml of ice-precooled sterile double distilled water;

5) centrifuging according to the step 3), and resuspending the thalli by using 20ml of ice-precooled 1M sorbitol solution;

6) centrifuging according to step 3), and resuspending the cells in 0.3ml of ice-precooled 1M sorbitol solution to a final volume of about 0.5 ml;

7) subpackaging into 80ul portions, and storing at-70 deg.C for use.

2.3 electrotransformation of Pichia pastoris

1) Washing the electric rotary cup with absolute ethyl alcohol for three times, and airing residual ethyl alcohol in a sterilized super clean bench;

2) sealing the electric rotary cup, and pre-cooling for 10min by rotating the electric rotary cup into ice;

3) thawing the pichia pastoris competent cells in ice pre-freezing, adding about 10ug of linearized pPIC9k-colI α 2 plasmid, gently mixing uniformly, transferring to a 0.2cm ice pre-cooling electric transfer cup, and continuously pre-cooling on ice for 5 min;

4) electric shock, wherein the voltage is 1.5kV, the capacitance is 25uF, the resistance is 200 omega, and the electric shock time is 5-10 mSec;

5) after the electric shock is finished, 1ml of ice-precooled 1M sorbitol solution is rapidly added, gently and uniformly blown and transferred to a 1.5ml centrifuge tube;

6) and (3) coating the bacterial suspension on an MD (MD) plate, coating one plate per 100-200 ul, standing for 10min at room temperature, and carrying out inverted culture at 30 ℃ for 2-5 days until a single bacterial colony appears.

2.4 screening of multicopy insertion recombinants

1) Adding 2ml of sterile double distilled water on the surface of the MD plate with the transformant growing, gently scraping the His + transformant on the surface of the MD plate by using a sterile triangular spreader, and transferring the His + transformant to a 50ml centrifuge tube;

2) adding 20ml of sterile double distilled water for dilution, uniformly mixing, and measuring the OD600 value (1OD600 is 5 multiplied by 107 cells/ml);

3) spreading 105 cells on YPD plate containing 0.5mg/ml G418, inverting, and culturing at 30 deg.C for 3-4 days;

4) 200ul YPD liquid medium was added to each well of a sterile 96-well plate;

5) inoculating transformants obtained on YPD plates containing 0.5mg/ml G418 to the step 4) by using sterile toothpicks, mixing uniformly, and culturing at 30 ℃ for 48 h;

6) after 48h, taking a new sterile 96-well plate, adding 190ul YPD liquid culture medium into each well, adding 10ul culture obtained by the first 96-well plate into the corresponding well, and culturing at 30 ℃ for 24 h;

7) after 24h, taking a new sterile 96-well plate, adding 190ul YPD liquid culture medium into each well, adding 10ul culture obtained by the second 96-well plate into the corresponding well, and culturing at 30 ℃ for 24 h;

8) after 24h, respectively taking out 1ul of the third 96-well plate, respectively spotting the obtained product on YPD plates containing 1.0mg/ml and 4mg/ml G418, and continuously culturing for 96 h-120 h at 30 ℃, if the Pichia pastoris SMD1168 transformant can grow on a culture medium containing higher concentration G418, the transformant contains more copies of the target gene, namely, a plurality of pPIC9k-colI α 2 fragments are integrated on a yeast chromosome, namely, the transformant has the potential of highly expressing the target protein.

3. Preparation of recombinant human collagen

1) YPD is used for carrying out overnight activation culture on the pichia pastoris engineering bacteria based on 220rpm at 30 ℃;

2) inoculating the bacterial liquid into a BMGY culture medium, culturing the engineering bacteria at 30 ℃ and 220rpm for 16-18h until OD600 is 2.0-6.0;

3) centrifuging at 1500g for 5min at room temperature, collecting thallus, suspending thallus with 10mL BMMY medium to OD600 of about 1.0, placing the obtained bacterial liquid in 100mL sterile triangular flask, and continuously performing shaking culture at 28 deg.C and 220 rpm;

4) adding methanol to the culture medium every 24 hours until the final concentration is 1% for induction culture;

5) centrifuging at 4 deg.C under 3000 Xg for 20min, and collecting supernatant; taking a small amount of supernatant to carry out SDS-PAGE analysis detection, and the result is shown in figure 3;

6) adding pure water with the volume of 5-7 times of that of the supernatant, and performing ultrafiltration concentration to 20% of the initial volume;

7) adding 100ul of 1 xNi-NTA binding buffer solution into 1ml of the concentrated solution, and fully mixing at 4 ℃;

8) adding 20ul of 50% Ni-NTA His band resin suspension, mixing gently, and combining for 30 min;

9) centrifuging at 15000 Xg for 10 s to precipitate the resin, and discarding the supernatant;

10) the resin was rinsed with 100ul of 1 XNi-NTA rinse buffer, centrifuged at 15000 Xg for 10 seconds, and the supernatant carefully aspirated. Repeating the steps again;

11) eluting the target protein with 200ul of 1 XNi-NTA elution buffer, centrifuging at 15000 Xg for 10 seconds, carefully transferring the supernatant to a clean vial, and repeating for 2 more times;

12) the supernatant was collected and dialyzed overnight at 4 ℃ using Anti-Flag affinity purification gel binding buffer;

13) adding 100ul Anti-Flag affinity purification gel suspension into the dialysate, gently mixing, and combining for 30 min;

14) centrifuging at 15000 Xg for 10 s to precipitate gel, and discarding the supernatant;

15) rinse the gel with 200ul 1 × Wash solution, centrifuge for 10 seconds at 15000 × g, carefully aspirate the supernatant and repeat again;

16) elute the protein of interest with 200ul 1 × Elution buffer, centrifuge at 15000 × g for 10 seconds, carefully transfer the supernatant to a clean vial, repeat 2 more times;

17) desalting and concentrating the obtained eluent, and performing vacuum freeze drying on the prepared concentrated solution to obtain the yeast recombinant collagen.

18) Pouring the concentrated solution into a glass culture dish, freezing overnight in a refrigerator at-20 deg.C, transferring into a freeze dryer precooled to-45 deg.C, starting a vacuum pump, and maintaining for 48 hr;

19) after the freeze-drying is finished, the air release valve is opened carefully until the internal air pressure and the external air pressure are balanced. And taking out the culture dish to obtain a white recombinant human collagen solid.

EXAMPLE two identification of recombinant human collagen

Examples 1A,

1. Western blotting (Western blotting) assay

1) Taking the fermented supernatant to carry out SDS-PAGE electrophoresis until bromophenol blue completely runs out of the lower edge of the separation gel;

2) taking out the gel from the glass plate, and soaking the gel in a membrane transfer buffer solution;

3) soaking the PVDF membrane with the same size as the gel in absolute methanol for 10 seconds, transferring the PVDF membrane into a membrane transferring buffer solution, and continuously soaking for later use;

4) placing sponge, filter paper, a PVDF membrane, gel, filter paper and sponge which are soaked in a membrane transferring buffer solution in advance in a transferring clamp in sequence to ensure that no air bubbles exist between each layer;

5) placing the transfer clip into an electric rotary tank, wherein the film is close to the anode, the glue is close to the cathode, placing the electric rotary tank into an ice water bath, and transferring for 2h at 450 mA;

6) taking out the PVDF membrane, soaking the PVDF membrane in 5% skimmed milk powder prepared by TBS solution, and shaking at room temperature for 2 h; discarding the milk powder liquid, adding a primary antibody prepared by 5% milk powder liquid, and shaking at room temperature for 2 h;

7) pouring out the primary antibody solution, washing the membrane with TBS solution for three times, each time for 5 min;

8) soaking the membrane into a goat anti-mouse IgG antibody secondary antibody solution marked by horseradish peroxidase (HRP) prepared by 5% milk powder solution, and oscillating for 2h at room temperature;

9) pouring out the secondary antibody solution, washing the membrane with TBS solution for three times, each time for 5 min;

10) absorbing residual moisture on the dry film by using filter paper, uniformly coating TMB (Beyotime) color development liquid on the film, and standing for a moment in dark light;

11) and (5) absorbing residual moisture on the membrane by using filter paper, and photographing and storing.

2. Identification by mass spectrometry

1) Firstly, taking a protein sample for SDS-PAGE electrophoretic analysis;

2) staining with Coomassie brilliant blue R-250, decolorizing, cutting the target protein band with a clean blade along the direction of the target protein band at a width of 1mm, and delivering the sample to a Putai organism for identification.

3) ProtTech's ProtQuest software suite software was used to perform alignments against the UniProt proteinatabase and NCBI databases.

Example 2, detection of the overall length of the recombinant human collagen:

the parents and the purification markers carried at the two ends of the recombinant human collagen are utilized, and the Flag monoclonal antibody and the HRP-coupled 6His monoclonal antibody can be utilized for sandwich ELISA detection so as to qualitatively or/and quantitatively analyze the recombinant human collagen in a sample. The method comprises the following specific steps:

1) the mouse anti-Flag monoclonal antibody was diluted 1000-fold with a binding buffer (1 × PBS buffer), and 100 μ L/well of an antibody dilution was added to the ELISA plate at an antibody concentration of 0.5 μ g/mL;

2) covering the plate with plastic sealing film, and incubating at 37 deg.C for 2h, or incubating overnight at 4 deg.C;

3) inverting the ELISA plate, throwing out the antibody solution, adding 200 μ L of washing solution (PBS buffer solution containing 0.05% Tween-20) into each well, and rinsing for 3 times, shaking for 5min each time;

4) adding 200 mu L of blocking solution (washing solution added with 1% BSA) into each hole to block the non-specific binding sites on the ELISA plate;

5) covering the plate with plastic sealing film, and incubating at 37 deg.C for 2h, or incubating overnight at 4 deg.C;

6) discarding the confining liquid, adding 200 μ L of washing liquid into each well, and rinsing for 3 times, shaking for 5min each time;

7) adding 100 mu L of recombinant human collagen samples which are properly diluted by confining liquid into each hole;

8) covering the plate with plastic sealing film, and incubating at 37 deg.C for 2h, or incubating overnight at 4 deg.C;

9) discarding the sample solution, adding 200 μ L of washing solution into each well, and rinsing for 3 times, shaking for 5min each time;

10) diluting HRP-coupled 6XHis monoclonal antibody 1000 times by using a confining liquid, and adding 100 mu L of antibody diluent into each hole;

11) covering the plate with plastic sealing film, and incubating at 37 deg.C for 30 min;

12) discarding the HRP-antibody solution, adding 200 μ L of washing solution into each well, and rinsing for 3 times, shaking for 5min each time;

13) adding 100 mu L/hole TMB reagent into the ELISA plate by using a multi-channel pipette, and enabling the reagent to present a sufficiently dark color for about 10-15 min;

14) adding 100 mu L of stop solution (1mol/L hydrochloric acid) into each hole to stop the color reaction;

15) and (3) measuring the light absorption value of each hole under 450nm within 10min by using a microplate reader to analyze the recombinant human collagen in the sample.

The results are shown in the following table:

EXAMPLE III preparation of collagen skin care preparation for external use

Example 1 the collagen skin care product for external use uses purified water as a solvent, and comprises, by mass, 0.05% of a recombinant human collagen, 2% of a humectant, 2% of butylene glycol, 0.05% of sodium hyaluronate, 0.05% of xanthan gum, 0.1% of allantoin, 0.5% of panthenol, 0.3% of dipotassium glycyrrhizinate, 20.05% of EDTA & Na0.3% of β -dextran, 0.3% of lumba oil, 0.2% of sodium lactate, 0.5% of water-soluble azone, and 0.05% of silk peptide, wherein the humectant is glycerin.

The preparation steps are as follows:

① is prepared from recombinant human collagen 0.05%, humectant 2%, butanediol 2%, sodium hyaluronate 0.05%, xanthan gum 0.05%, allantoin 0.1%, panthenol 0.5%, dipotassium glycyrrhizinate 0.3%, EDTA & Na20.05%, β -dextran 0.3%, lumba oil 0.3%, sodium lactate 0.2%, water-soluble azone 0.5%, and silk peptide 0.05%;

② dissolving the raw materials in ① with purified water, and stirring to obtain colorless, odorless, transparent, and moisture-keeping essence.

Example 2 the collagen skin care product for external use uses purified water as a solvent, and comprises, by mass, 0.01% of recombinant human collagen, 1% of a humectant, 1% of butylene glycol, 0.1% of sodium hyaluronate, 0.1% of xanthan gum, 0.2% of allantoin, 1% of panthenol, 0.5% of dipotassium glycyrrhizinate, 20.1% of EDTA & Na20, 0.5% of β -dextran, 0.5% of lubrabar oil, 0.3% of sodium lactate, 1% of water-soluble azone, and 0.1% of silk peptide, wherein the humectant is glycerin.

The preparation steps are as follows:

① is prepared from recombinant human collagen 0.01%, humectant 1%, butanediol 1%, sodium hyaluronate 0.1%, xanthan gum 0.1%, allantoin 0.2%, panthenol 1%, dipotassium glycyrrhizinate 0.5%, EDTA & Na20.1%, β -dextran 0.5%, lumba oil 0.5%, sodium lactate 0.3%, water-soluble azone 1%, and silk peptide 0.1%, wherein the humectant is glycerol;

Example 3, the collagen skin care product for external use includes, in terms of mass percentages, 0.1% of a recombinant human collagen, 3% of a humectant, 3% of butylene glycol, 0.01% of sodium hyaluronate, 0.01% of xanthan gum, 0.01% of allantoin, 0.1% of panthenol, 0.1% of dipotassium glycyrrhizinate, 20.01% of EDTA & Na20, 0.1% of β -dextran, 0.1% of luba oil, 0.1% of sodium lactate, 0.1% of water-soluble azone, and 0.01% of silk peptide, wherein the humectant is glycerin.

The preparation steps are as follows:

① is prepared from recombinant human collagen 0.1%, humectant 3%, butylene glycol 3%, sodium hyaluronate 0.01%, xanthan gum 0.01%, allantoin 0.01%, panthenol 0.1%, dipotassium glycyrrhizinate 0.1%, EDTA & Na20.01%, β -dextran 0.1%, lumba oil 0.1%, sodium lactate 0.1%, water-soluble azone 0.1%, and silk peptide 0.01%, wherein the humectant is glycerol;

The use method of the recombinant human collagen skin care product for external use comprises the following steps: after cleaning the face in the morning and evening, the face lotion is directly smeared on the face and gently flicked until the face lotion is completely absorbed.

Example four preparation examples of recombinant human collagen applied to introduced skin care products

Example 1, the mass fractions of the components of the skin care product for introduction are as follows: 0.1% of recombinant human collagen; 1% of a humectant; 0.1% of sodium hyaluronate; 0.05% of micromolecular sodium hyaluronate; 0.9 percent of sodium chloride; the balance being purified water.

The preparation steps are as follows:

① is prepared by weighing 0.1% of recombinant human collagen, 1% of humectant, 0.1% of sodium hyaluronate, 0.05% of small molecule sodium hyaluronate, 0.9% of sodium chloride and the balance of purified water, wherein the humectant is glycerol, accurately weighing each component, fully stirring and mixing uniformly, and fixing the volume;

② filtering with 0.22um microporous membrane for sterilization;

③ and packaging into disposable sterile syringes, each syringe is filled with 3ml under sterile condition, and packaging under sterile condition.

④ when used, it can be directly taken out and used in conjunction with an introduction instrument.

Example 2, the mass fractions of the components of the skin care product for introduction are as follows: 0.5 percent of recombinant human collagen; 10% of a humectant; 0.5 percent of sodium hyaluronate; 0.01 percent of micromolecular sodium hyaluronate; 0.9 percent of sodium chloride; the balance being purified water.

The preparation steps are as follows:

① is prepared by weighing 0.5% of recombinant human collagen, 10% of humectant, 0.5% of sodium hyaluronate, 0.01% of small molecule sodium hyaluronate, 0.9% of sodium chloride and the balance of purified water, wherein the humectant is glycerol, accurately weighing each component, fully stirring and mixing uniformly, and fixing the volume;

② filtering with 0.22um microporous membrane for sterilization;

Example 3, the mass fractions of the components of the skin care product for introduction are as follows: 0.3 percent of recombinant human collagen; 5% of a humectant; 0.3 percent of sodium hyaluronate; 0.03 percent of micromolecular sodium hyaluronate; 0.9 percent of sodium chloride; the balance being purified water.

The preparation steps are as follows:

① is prepared by weighing 0.3% of recombinant human collagen, 5% of humectant, 0.3% of sodium hyaluronate, 0.03% of small molecule sodium hyaluronate, 0.9% of sodium chloride and the balance of purified water, wherein the humectant is glycerol, accurately weighing each component, fully stirring and uniformly mixing, and fixing the volume;

② filtering with 0.22um microporous membrane for sterilization;

EXAMPLES five examples of preparation of collagen gel dressings

The recombinant human collagen is used for preparing the original protein plaster, the collagen gel takes purified water as a solvent, and the components of the collagen gel also comprise yeast recombinant collagen, a gelling agent and a humectant; the gel is carbomer; the humectant is glycerol and triethanolamine;

example 1, the mass ratio of each component is as follows: 0.1% of recombinant human collagen, 1% of glycerol, 4% of carbomer and 10% of triethanolamine.

The preparation steps are as follows:

① weighing appropriate amount of glycerol and carbomer, stirring for 1 hr, and dissolving completely;

② adding purified water, weighing appropriate amount of recombinant human collagen, stirring for 1 hr, and dissolving completely;

③ adding triethanolamine, stirring for 1 hr, and mixing to obtain collagen gel dressing product.

Example 2, the mass ratio of each component is as follows: 0.5% of recombinant human collagen, 10% of glycerol, 0.1% of carbomer and 0.1% of triethanolamine.

The preparation steps are as follows:

Example 3, the mass ratio of each component is as follows: 0.3% of recombinant human collagen, 5% of glycerol, 2% of carbomer and 5% of triethanolamine.

The preparation steps are as follows:

EXAMPLES preparation of a Hexacollagen gel dressing

The recombinant human-derived collagen is used for preparing the original protein dressing and consists of the recombinant human-derived collagen, a humectant, a medical thickening agent and a non-woven fabric base material, wherein the humectant is glycerol, and the medical thickening agent is xanthan gum.

Example 1, the mass ratio of each component is as follows: 0.5% of recombinant human collagen, 10% of glycerol, 0.1% of xanthan gum and the balance of purified water.

The preparation steps are as follows:

① weighing the components accurately, dissolving in purified water, stirring, and mixing;

② and quantitatively filling into medical aluminum foil bags containing non-woven fabric base materials, sealing, and performing irradiation sterilization to obtain the collagen patch product.

Example 2, the mass ratio of each component is as follows: 0.1% of recombinant human collagen, 1% of glycerol, 2% of xanthan gum and the balance of purified water.

The preparation steps are as follows:

Example 3, the mass ratio of each component is as follows: 0.3% of recombinant human collagen, 5% of glycerol, 1% of xanthan gum and the balance of purified water.

The preparation steps are as follows:

EXAMPLES preparation of seven collagen-based dressings for skin and mucous membrane protection

Example 1, the collagen-based skin mucosa protective agent dressing comprises the following components in percentage by mass: 0.1% of recombinant human collagen, 0.1% of sodium hyaluronate and 40% of glycerol; sterilizing the prepared preparation solution, and aseptically filling.

The preparation steps are as follows:

① weighing appropriate amount of each component, dissolving in purified water, and stirring for 1 hr;

② weighing recombinant human collagen, supplementing purified water, stirring, dissolving completely, and mixing;

③ sterile filling, sealing, and sterilizing by irradiation to obtain collagen-based skin mucosa protective agent dressing for skin mucosa protection after micro-plastic.

Example 2, the collagen-based skin mucosa protective agent dressing comprises the following components in percentage by mass: 1% of recombinant human collagen and 1% of sodium hyaluronate; sterilizing the prepared preparation solution, and aseptically filling.

The preparation steps are as follows:

Example 3, the collagen-based skin mucosa protective agent dressing comprises the following components in percentage by mass: 0.5% of recombinant human collagen, 0.5% of sodium hyaluronate and 20% of glycerol; sterilizing the prepared preparation solution, and aseptically filling.

The preparation steps are as follows:

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

SEQUENCE LISTING

110 Jiangsu Zhizhi biomedicine Limited

< 120 > recombinant human collagen and application thereof

〈130〉UCFI2H

〈160〉

〈170〉

Amino acid sequence of recombinant human collagen of SEQ ID NO.1

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

SEQ ID NO.2 human type I collagen α 2 chain mature peptide gene DNA sequence

CAATACGACGGTAAAGGTGTTGGACTGGGTCCAGGTCCAATGGGTTTGATGGGACCTAGAGGTCCACCTGGAGCAGCTGGTGCCCCAGGACCTCAGGGTTTCCAAGGACCAGCAGGAGAGCCAGGAGAACCAGGTCAAACAGGACCAGCAGGTGCAAGAGGACCCGCAGGTCCCCCTGGAAAAGCAGGTGAAGACGGTCATCCAGGAAAACCCGGTAGACCTGGAGAAAGAGGTGTCGTAGGTCCCCAAGGTGCAAGAGGATTTCCCGGAACTCCAGGATTACCAGGTTTTAAGGGAATTAGAGGACATAACGGATTAGATGGACTGAAAGGACAACCTGGTGCACCAGGAGTTAAGGGAGAGCCTGGAGCACCAGGAGAAAACGGAACTCCAGGACAAACAGGTGCAAGAGGTCTGCCTGGAGAGAGAGGAAGAGTTGGAGCCCCAGGTCCCGCAGGTGCAAGAGGTTCAGACGGATCTGTCGGACCTGTGGGTCCAGCAGGTCCCATAGGTTCCGCAGGACCACCAGGATTCCCCGGAGCACCCGGACCAAAAGGAGAGATCGGAGCAGTCGGTAACGCAGGACCTGCAGGACCTGCCGGTCCTAGAGGAGAAGTTGGTTTACCAGGATTGTCAGGACCCGTGGGTCCACCTGGTAATCCAGGTGCCAACGGTTTAACAGGAGCAAAGGGAGCCGCAGGATTACCAGGTGTTGCAGGAGCACCAGGACTGCCAGGACCAAGAGGTATTCCTGGACCTGTTGGTGCAGCCGGTGCAACAGGTGCCAGAGGATTAGTAGGAGAACCAGGTCCAGCTGGTTCAAAAGGAGAGTCAGGAAACAAAGGAGAACCAGGTTCCGCAGGTCCACAGGGTCCTCCAGGTCCTTCAGGAGAAGAGGGAAAAAGAGGACCCAATGGAGAGGCAGGTTCAGCAGGACCCCCGGGTCCCCCAGGTCTGAGAGGATCACCCGGTAGTAGAGGTCTTCCAGGAGCAGACGGTAGAGCAGGTGTCATGGGTCCACCAGGATCAAGAGGTGCATCCGGTCCAGCTGGTGTAAGAGGACCAAATGGAGATGCAGGAAGACCAGGTGAGCCAGGACTTATGGGTCCTAGAGGTCTTCCAGGATCTCCCGGAAATATCGGACCCGCAGGAAAGGAGGGTCCAGTTGGTTTACCAGGTATCGACGGAAGACCTGGACCAATCGGACCAGCTGGAGCAAGAGGTGAGCCAGGAAATATCGGATTTCCCGGTCCAAAGGGTCCCACCGGAGATCCTGGAAAGAATGGTGACAAAGGTCACGCAGGTTTAGCAGGTGCAAGAGGTGCCCCCGGACCAGATGGAAACAACGGTGCCCAGGGTCCACCTGGTCCCCAGGGTGTGCAGGGAGGTAAAGGAGAGCAAGGACCACCAGGACCTCCTGGTTTCCAAGGATTACCCGGACCAAGTGGTCCAGCTGGTGAGGTGGGAAAACCAGGTGAGAGAGGTCTGCATGGAGAGTTCGGTCTTCCAGGTCCAGCCGGACCCAGAGGAGAGAGAGGTCCCCCTGGTGAATCCGGTGCCGCCGGTCCCACAGGTCCCATCGGAAGTAGAGGTCCATCCGGTCCTCCCGGACCCGACGGAAACAAAGGTGAACCAGGAGTGGTTGGTGCTGTCGGAACCGCTGGACCATCCGGTCCCTCAGGACTGCCTGGTGAAAGAGGTGCAGCTGGAATCCCTGGTGGAAAGGGAGAGAAAGGAGAACCCGGACTGAGAGGTGAAATAGGTAACCCAGGAAGAGATGGTGCCAGAGGTGCTCCGGGTGCTGTGGGTGCCCCTGGACCCGCCGGTGCTACCGGAGACAGAGGAGAAGCCGGAGCAGCCGGTCCAGCAGGACCCGCAGGACCCAGAGGTTCCCCTGGAGAGAGAGGAGAGGTTGGTCCTGCAGGTCCCAACGGTTTTGCCGGACCTGCCGGAGCTGCAGGACAGCCAGGAGCAAAAGGAGAAAGAGGTGCAAAGGGTCCTAAAGGTGAAAATGGTGTTGTGGGACCAACAGGTCCAGTCGGAGCCGCTGGTCCTGCAGGACCCAATGGTCCCCCTGGACCTGCTGGTTCAAGAGGAGATGGTGGACCACCAGGTATGACCGGATTTCCTGGTGCAGCTGGAAGAACAGGTCCTCCAGGTCCATCAGGAATCAGTGGTCCACCAGGTCCTCCTGGACCCGCAGGAAAAGAGGGTTTAAGAGGACCCAGAGGTGACCAAGGACCTGTTGGTAGAACTGGTGAGGTGGGTGCAGTCGGTCCACCTGGTTTTGCAGGAGAGAAGGGACCATCCGGTGAGGCAGGAACAGCAGGACCACCAGGTACTCCTGGTCCACAAGGATTATTAGGAGCCCCAGGTATTCTGGGTCTGCCAGGATCAAGAGGAGAGAGAGGACTTCCAGGTGTTGCCGGAGCCGTAGGAGAACCAGGACCCCTGGGTATAGCTGGACCTCCCGGTGCTAGAGGACCTCCTGGTGCCGTAGGTTCCCCAGGTGTGAATGGTGCTCCAGGAGAGGCAGGAAGAGATGGTAACCCCGGTAATGACGGACCACCAGGTAGAGACGGACAACCAGGTCATAAAGGTGAAAGAGGATACCCAGGTAACATCGGTCCTGTTGGAGCAGCCGGAGCCCCCGGACCTCATGGACCAGTCGGACCCGCCGGTAAACATGGTAACAGAGGAGAGACCGGACCTAGTGGACCTGTCGGACCCGCAGGTGCAGTTGGACCTAGAGGACCCTCCGGTCCCCAGGGTATAAGAGGAGACAAGGGTGAACCAGGAGAAAAGGGACCAAGAGGTCTGCCAGGTTTAAAAGGTCATAATGGATTACAGGGACTGCCAGGAATAGCAGGACATCATGGAGACCAGGGTGCTCCAGGTTCAGTTGGACCAGCAGGTCCAAGAGGACCAGCAGGACCAAGTGGACCCGCAGGAAAGGATGGAAGAACTGGTCATCCAGGAACTGTTGGACCAGCAGGAATTAGAGGACCACAAGGTCACCAGGGACCCGCTGGACCACCTGGTCCCCCAGGACCTCCCGGACCTCCCGGTGTGTCCGGTGGAGGTTACGATTTTGGTTATGACGGAGACTTTTACAGAGCA

Primer DNA sequence P1 of SEQ ID NO.3

GACTGGTTCCAATTGACAAGC

Primer DNA sequence P2 of SEQ ID NO.4

GGGCCCCTTGTCATCGTCATCCTTGTAGTCTCTTTTCTCGAGAGATAC

Primer DNA sequence P3 of SEQ ID NO.5

GACTACAAGGATGACGATGACAAGGGGCCCCAATACGACGGTAAAGGTGTTGGACTG

Primer DNA sequence P4 of SEQ ID NO.6

GCTCTGTAAAAGTCTCCGTCA

Primer DNA sequence P5 of SEQ ID NO.7

GACGGAGACTTTTACAGAGCACACCATCACCATCATCACTAAC

Primer DNA sequence P6 of SEQ ID NO.8

AATTAATTCGCGGCCGCCCTAGGTTAGTGATGATGGTG

Gene DNA sequence corresponding to recombinant human collagen of SEQ ID NO.9

GACTACAAGGATGACGATGACAAGGGGCCCCAATACGACGGTAAAGGTGTTGGACTGGGTCCAGGTCCAATGGGTTTGATGGGACCTAGAGGTCCACCTGGAGCAGCTGGTGCCCCAGGACCTCAGGGTTTCCAAGGACCAGCAGGAGAGCCAGGAGAACCAGGTCAAACAGGACCAGCAGGTGCAAGAGGACCCGCAGGTCCCCCTGGAAAAGCAGGTGAAGACGGTCATCCAGGAAAACCCGGTAGACCTGGAGAAAGAGGTGTCGTAGGTCCCCAAGGTGCAAGAGGATTTCCCGGAACTCCAGGATTACCAGGTTTTAAGGGAATTAGAGGACATAACGGATTAGATGGACTGAAAGGACAACCTGGTGCACCAGGAGTTAAGGGAGAGCCTGGAGCACCAGGAGAAAACGGAACTCCAGGACAAACAGGTGCAAGAGGTCTGCCTGGAGAGAGAGGAAGAGTTGGAGCCCCAGGTCCCGCAGGTGCAAGAGGTTCAGACGGATCTGTCGGACCTGTGGGTCCAGCAGGTCCCATAGGTTCCGCAGGACCACCAGGATTCCCCGGAGCACCCGGACCAAAAGGAGAGATCGGAGCAGTCGGTAACGCAGGACCTGCAGGACCTGCCGGTCCTAGAGGAGAAGTTGGTTTACCAGGATTGTCAGGACCCGTGGGTCCACCTGGTAATCCAGGTGCCAACGGTTTAACAGGAGCAAAGGGAGCCGCAGGATTACCAGGTGTTGCAGGAGCACCAGGACTGCCAGGACCAAGAGGTATTCCTGGACCTGTTGGTGCAGCCGGTGCAACAGGTGCCAGAGGATTAGTAGGAGAACCAGGTCCAGCTGGTTCAAAAGGAGAGTCAGGAAACAAAGGAGAACCAGGTTCCGCAGGTCCACAGGGTCCTCCAGGTCCTTCAGGAGAAGAGGGAAAAAGAGGACCCAATGGAGAGGCAGGTTCAGCAGGACCCCCGGGTCCCCCAGGTCTGAGAGGATCACCCGGTAGTAGAGGTCTTCCAGGAGCAGACGGTAGAGCAGGTGTCATGGGTCCACCAGGATCAAGAGGTGCATCCGGTCCAGCTGGTGTAAGAGGACCAAATGGAGATGCAGGAAGACCAGGTGAGCCAGGACTTATGGGTCCTAGAGGTCTTCCAGGATCTCCCGGAAATATCGGACCCGCAGGAAAGGAGGGTCCAGTTGGTTTACCAGGTATCGACGGAAGACCTGGACCAATCGGACCAGCTGGAGCAAGAGGTGAGCCAGGAAATATCGGATTTCCCGGTCCAAAGGGTCCCACCGGAGATCCTGGAAAGAATGGTGACAAAGGTCACGCAGGTTTAGCAGGTGCAAGAGGTGCCCCCGGACCAGATGGAAACAACGGTGCCCAGGGTCCACCTGGTCCCCAGGGTGTGCAGGGAGGTAAAGGAGAGCAAGGACCACCAGGACCTCCTGGTTTCCAAGGATTACCCGGACCAAGTGGTCCAGCTGGTGAGGTGGGAAAACCAGGTGAGAGAGGTCTGCATGGAGAGTTCGGTCTTCCAGGTCCAGCCGGACCCAGAGGAGAGAGAGGTCCCCCTGGTGAATCCGGTGCCGCCGGTCCCACAGGTCCCATCGGAAGTAGAGGTCCATCCGGTCCTCCCGGACCCGACGGAAACAAAGGTGAACCAGGAGTGGTTGGTGCTGTCGGAACCGCTGGACCATCCGGTCCCTCAGGACTGCCTGGTGAAAGAGGTGCAGCTGGAATCCCTGGTGGAAAGGGAGAGAAAGGAGAACCCGGACTGAGAGGTGAAATAGGTAACCCAGGAAGAGATGGTGCCAGAGGTGCTCCGGGTGCTGTGGGTGCCCCTGGACCCGCCGGTGCTACCGGAGACAGAGGAGAAGCCGGAGCAGCCGGTCCAGCAGGACCCGCAGGACCCAGAGGTTCCCCTGGAGAGAGAGGAGAGGTTGGTCCTGCAGGTCCCAACGGTTTTGCCGGACCTGCCGGAGCTGCAGGACAGCCAGGAGCAAAAGGAGAAAGAGGTGCAAAGGGTCCTAAAGGTGAAAATGGTGTTGTGGGACCAACAGGTCCAGTCGGAGCCGCTGGTCCTGCAGGACCCAATGGTCCCCCTGGACCTGCTGGTTCAAGAGGAGATGGTGGACCACCAGGTATGACCGGATTTCCTGGTGCAGCTGGAAGAACAGGTCCTCCAGGTCCATCAGGAATCAGTGGTCCACCAGGTCCTCCTGGACCCGCAGGAAAAGAGGGTTTAAGAGGACCCAGAGGTGACCAAGGACCTGTTGGTAGAACTGGTGAGGTGGGTGCAGTCGGTCCACCTGGTTTTGCAGGAGAGAAGGGACCATCCGGTGAGGCAGGAACAGCAGGACCACCAGGTACTCCTGGTCCACAAGGATTATTAGGAGCCCCAGGTATTCTGGGTCTGCCAGGATCAAGAGGAGAGAGAGGACTTCCAGGTGTTGCCGGAGCCGTAGGAGAACCAGGACCCCTGGGTATAGCTGGACCTCCCGGTGCTAGAGGACCTCCTGGTGCCGTAGGTTCCCCAGGTGTGAATGGTGCTCCAGGAGAGGCAGGAAGAGATGGTAACCCCGGTAATGACGGACCACCAGGTAGAGACGGACAACCAGGTCATAAAGGTGAAAGAGGATACCCAGGTAACATCGGTCCTGTTGGAGCAGCCGGAGCCCCCGGACCTCATGGACCAGTCGGACCCGCCGGTAAACATGGTAACAGAGGAGAGACCGGACCTAGTGGACCTGTCGGACCCGCAGGTGCAGTTGGACCTAGAGGACCCTCCGGTCCCCAGGGTATAAGAGGAGACAAGGGTGAACCAGGAGAAAAGGGACCAAGAGGTCTGCCAGGTTTAAAAGGTCATAATGGATTACAGGGACTGCCAGGAATAGCAGGACATCATGGAGACCAGGGTGCTCCAGGTTCAGTTGGACCAGCAGGTCCAAGAGGACCAGCAGGACCAAGTGGACCCGCAGGAAAGGATGGAAGAACTGGTCATCCAGGAACTGTTGGACCAGCAGGAATTAGAGGACCACAAGGTCACCAGGGACCCGCTGGACCACCTGGTCCCCCAGGACCTCCCGGACCTCCCGGTGTGTCCGGTGGAGGTTACGATTTTGGTTATGACGGAGACTTTTACAGAGCACACCATCACCATCATCACTAA

Primer DNA sequence P7 of SEQ ID NO.10

GCCGGTAAACATGGTAACAGA

Primer DNA sequence P8 of SEQ ID NO.11

GCAAATGGCATTCTGACATCC

Claims

1. The recombinant human collagen is characterized in that 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 type I collagen α 2 chain mature peptide chain and a carboxyl-terminal affinity purification tag from an amino terminal.

2. The recombinant human collagen according to claim 1, wherein said amino-terminal affinity purification tag is Flag tag and said carboxy-terminal affinity purification tag is 6His tag.

3. The recombinant human collagen according to claim 1, wherein the recombinant human collagen has a total length of 1056 amino acids.

4. The recombinant human collagen according to claim 1, wherein the gene sequence of the recombinant human collagen is as shown in SEQ ID NO: shown at 9.

5. The recombinant human collagen according to claim 1, wherein the gene sequence of the mature peptide chain of human type I collagen α 2 is obtained by adjusting the codon usage frequency of the codon according to the host codon with reference to the native collagen gene sequence.

6. The recombinant human collagen according to claim 5, wherein said host is Pichia pastoris and said modifications are to replace yeast unusual codons in the native collagen gene sequence with their customary codons.

7. The recombinant human collagen of claim 4, wherein the recombinant human collagen is amplified by introducing a gene sequence encoding a Flag tag sequence at the 5 'end and introducing a gene sequence encoding a 6His sequence at the 3' end of the collagen gene sequence obtained in claim 5 during primer amplification construction.

8. The recombinant human collagen according to claim 6, wherein said recombinant human collagen is extracted and purified from fermentation broth of Pichia pastoris by Ni-NTA His Bind resin and Anti-Flag affinity purification gel adsorption method.

9. The application of the recombinant human collagen is characterized in that the recombinant human collagen is applied to an external skin care product, and the external skin care product comprises the following components in parts by mass:

the recombinant human collagen of any one of claims 1-8 at a concentration of 0.01% to 0.1%;

1% -3% of a humectant;

1% -3% of butanediol;

0.01 to 0.1 percent of sodium hyaluronate;

0.01 to 0.1 percent of xanthan gum;

0.01 to 0.2 percent of allantoin;

0.1 to 1 percent of panthenol;

0.1 to 0.5 percent of dipotassium glycyrrhizinate;

EDTA·Na2 0.01％-0.1％；

β -dextran 0.1% -0.5%;

0.1 to 0.5 percent of luba oil;

sodium lactate 0.1-0.3%;

0.1 to 1 percent of water-soluble azone;

0.01% -0.1% of silk peptide;

the balance being purified water.

10. The application of the recombinant human collagen is characterized in that the recombinant human collagen is applied to an introduced skin care product, and the introduced skin care product comprises the following components in percentage by mass:

the recombinant human collagen of any one of claims 1-8 from 0.1% to 0.5%;

1% -10% of humectant;

0.1 to 0.5 percent of sodium hyaluronate;

0.01-0.05% of micromolecular sodium hyaluronate;

0.9 percent of sodium chloride;

the balance being purified water.

11. The application of the recombinant human collagen is characterized in that the recombinant human collagen is applied to preparation of a collagen gel dressing, and the collagen gel dressing comprises the following components in parts by mass:

0.1-0.5% of the recombinant human collagen according to any one of claims 1-8;

1% -10% of humectant;

0.1% -4% of carbomer;

0.1 to 10 percent of triethanolamine;

the balance being purified water.

12. The application of the recombinant human collagen is characterized in that the recombinant human collagen is used for preparing a raw protein plaster dressing, and the raw protein plaster dressing comprises the following components in parts by mass:

0.1-0.5% of the recombinant human collagen according to any one of claims 1-8;

1% -10% of humectant;

0.1% -2% of xanthan gum;

the balance being purified water.

13. The application of the recombinant human collagen is characterized in that the recombinant human collagen is applied to preparation of a collagen-based skin mucosa protective agent dressing, and the collagen-based skin mucosa protective agent dressing comprises the following components in parts by mass:

0.1% -1% of the recombinant human collagen according to any one of claims 1-8;

0.1 to 1 percent of sodium hyaluronate;

0-40% of a humectant;

the balance being purified water.