CN114874316A

CN114874316A - Water-soluble collagen and application thereof

Info

Publication number: CN114874316A
Application number: CN202210727255.2A
Authority: CN
Inventors: 孙琰奇; 杨清雄; 王伯初; 郝石磊; 祝连彩
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2022-08-09
Anticipated expiration: 2042-06-24
Also published as: CN114874316B

Abstract

The invention belongs to the field of biological medicines, and particularly relates to water-soluble collagen and application thereof. The invention provides a water-soluble collagen, which comprises an alpha 1 chain and an alpha 2 chain; the amino acid sequence of the alpha 1 chain is shown as SEQ ID NO. 1; the amino acid sequence of the alpha 2 chain is shown as SEQ ID NO. 2. The water-soluble collagen has good water solubility and obvious anti-fatigue effect, and has good application prospect in the field of biological medicine.

Description

Water-soluble collagen and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to water-soluble collagen and application thereof.

Background

Collagen (Collagen) is a kind of protein with the largest content in mammals, widely distributed in animal skin, bones, tendons, ligaments and blood vessels, has important functions of protecting organisms and supporting organs, and is an important component of extracellular matrix. The collagen is rich in various amino acids needed by human bodies, and has wide application in the fields of medicines and foods. Collagen is mostly a triple helix formed by winding three polypeptide chains. Collagen exists in different types due to the combination of its three alpha helical chains. The type I collagen is composed of 2 alpha 1 chains and 1 alpha 2 chain which are cross-linked.

Natural collagen is insoluble in water, and drugs on the market, which mainly contain collagen, have very low solubility in water and non-uniform properties, and are difficult to be fully utilized by the human body. In addition, collagen taken directly from animal tissue is difficult to avoid viral infection and other problems. Therefore, these reasons make the relevant drugs difficult to be effectively absorbed and utilized by the body, and the application range of the drugs is limited, so that the biological functions of collagen cannot be fully exerted.

In recent years, to solve the problem of limited use of traditional animal collagen, many scholars and enterprises have begun to use biotechnology to obtain recombinant collagen by means of in vitro recombinant expression. Genetic engineering techniques for collagen synthesis using microorganisms have become more and more mature. The recombinant collagen not only well retains the advantages of natural collagen, but also has the advantages of no limitation on material sources, low cost and the like, and is widely applied to a plurality of fields of health care products, cosmetics, wound repair and the like. However, an expression system using bacteria such as escherichia coli as an expression host contains endotoxin, pyrogen and the like, and thus, has a biological potential safety hazard; the expressed protein exists in the host cell in the form of inclusion body, which is not beneficial to the purification and recovery of the product; prokaryotic expression systems are relatively low in grade, and cannot complete post-translational modification of the expression product and render the expression product biologically active. In contrast, pichia pastoris has a great advantage. Pichia pastoris can be used for secretion expression of foreign proteins, is beneficial to separation and purification of proteins in downstream production, does not have biological safety problems such as endotoxin and pyrogen like bacteria, and is widely used in various fields such as food and medicines.

Therefore, there is a need to design a water-soluble collagen and express it by eukaryotic microorganisms to improve the utilization and bioactivity of collagen.

Disclosure of Invention

The invention aims to solve the problems of low utilization rate of natural collagen, potential safety hazard in the production of the collagen by a prokaryotic expression system, complex product purification process and low product bioactivity.

In order to solve the above problems, the present invention provides a water-soluble collagen comprising an α 1 chain and an α 2 chain; the alpha 1 chain is named as Col1 alpha 1, and the amino acid sequence of the alpha 1 chain is shown as SEQ ID NO: 1; the alpha 2 chain is named as Col1 alpha 2, and the amino acid sequence of the alpha 2 chain is shown in SEQ ID NO. 2.

In the water-soluble collagen, the molar ratio of the α 1 chain to the α 2 chain may be 2: 1.

The invention also provides a coding gene of the water-soluble collagen.

In the coding gene, the coding gene sequence of the alpha 1 chain can be SEQ ID NO. 3; the coding gene sequence of the alpha 2 chain can be SEQ ID NO. 4.

Expression cassettes, vectors or host bacteria containing the above-described encoding genes also fall within the scope of the present invention.

The vector may be pPIC 9K. The recombinant vectors containing the coding gene of the water-soluble collagen are pPIC9K-Col1 alpha 1 and pPIC9K-Col1 alpha 2. The pPIC9K-Col1 alpha 1 expresses the alpha 1 chain of the water-soluble collagen, and the pPIC9K-Col1 alpha 2 expresses the alpha 2 chain of the water-soluble collagen. The coding gene of the alpha 1 chain and the coding gene of the alpha 2 chain can be introduced between the SnaB I site and the Avr II site of the pPIC9K vector through enzyme digestion and connection reaction.

The host bacterium may be a cloning host bacterium or an expression host bacterium. The cloning host bacterium may be E.coli (Escherichia coli) strain DH5 alpha. The expression host bacterium may be Pichia pastoris (Pichia pastoris). The pPIC9K-Col1 alpha 1 and the pPIC9K-Col1 alpha 2 can be introduced into Pichia pastoris by the electroporation method. The pPIC9K-Col1 alpha 1 and the pPIC9K-Col1 alpha 2 can be cut by restriction enzyme Sac I to obtain linearized vectors before electric shock transformation.

The invention also provides a method for preparing the water-soluble collagen, which comprises the following steps: and (3) introducing the coding gene of the water-soluble collagen into an expression host bacterium to obtain a recombinant bacterium, culturing the recombinant bacterium, inducing protein expression, and extracting and purifying the protein.

The expression host bacterium may be Pichia pastoris (Pichia pastoris).

In the method, recombinant vectors pPIC9K-Col1 alpha 1 and pPIC9K-Col1 alpha 2 can be respectively constructed and are respectively introduced into Pichia pastoris to obtain recombinant Pichia pastoris Picia-pPIC 9K-Col1 alpha 1 and Pichia-pPIC9K-Col1 alpha 2; separately culturing Pichia-pPIC9K-Col1 α 1 and Pichia-pPIC9K-Col1 α 2, adding methanol to the culture medium to induce protein expression, and collecting the supernatant; purifying the protein in the supernatant by trichloroacetic acid precipitation to obtain an alpha 1 chain and an alpha 2 chain of the water-soluble collagen; when the collagen is used, the alpha 1 chain and the alpha 2 chain are mixed according to the molar ratio of 2:1 to obtain the water-soluble collagen.

The invention also provides an anti-fatigue product, which comprises the water-soluble collagen.

The product can be prepared into capsules, powder, tablets, granules, pills, emulsions, pastes or solutions.

The administration mode of the product can be oral, injection, instillation or external application.

The application of the water-soluble collagen in preparing the anti-fatigue product also belongs to the protection scope of the invention.

The invention obtains the collagen with good water solubility through protein design, whole gene synthesis, vector construction and pichia pastoris expression. In a mouse weight swimming experiment, compared with a blank group, the water-soluble collagen prolongs the weight swimming time of the mouse by about 50 percent; in the mouse pole climbing experiment, compared with a blank group, the water-soluble collagen provided by the invention prolongs the pole climbing time of the mouse by nearly 1 time. Therefore, the water-soluble collagen can be effectively absorbed and utilized by organisms, has a remarkable anti-fatigue effect, and has a good application prospect in the field of biomedicine.

Drawings

FIG. 1 shows the results of hydrophobicity analysis of the α 1 chain (Col1 α 1) of the water-soluble collagen of the present invention. The abscissa is the amino acid position; the ordinate is the hydrophobicity evaluation score.

FIG. 2 shows the results of hydrophobicity analysis of the α 2 chain (Col1 α 2) of the water-soluble collagen of the present invention. The abscissa is the amino acid position; the ordinate is the hydrophobicity evaluation score.

FIG. 3 shows SDS-PAGE of expressed and purified Col1 α 1 and Col1 α 2.

FIG. 4 is a graph showing the results of a mouse weight swimming test. The abscissa is the group; the ordinate represents the weight bearing swimming time (min) of the mouse.

FIG. 5 is a graph showing the results of the pole climbing experiment of mice. The abscissa is the group; the ordinate is the mouse pole-climbing time (min).

Detailed Description

The present invention is further illustrated below by reference to examples, which are to be understood as being illustrative and illustrative only and not limiting in any way to the scope of the present invention.

Bacterial strains

Coli DH 5. alpha. competent cells were purchased from Beijing Solebao technologies, Inc. Pichia pastoris GS115 competent cells were purchased from Invitrogen.

Carrier

The pPIC9K plasmid is a Pichia expression vector, purchased from Invitrogen. The promoter of the plasmid is AOX1, the size of the vector is 9276bp, and the resistance of the vector is Ampicillin (Ampicillin) and Kanamycin (Kanamycin). The plasmid utilizes alpha factor to secrete signal peptide and secrete and express protein gene.

The MD solid medium, YPD solid medium, BMGY medium and BMMY medium used in the following examples were all prepared in the laboratory and have the following formulations:

MD solid medium: weighing 15g of agar powder in 860ml of distilled water, autoclaving for 121-20 min, cooling to about 60 ℃, and sequentially adding: 100ml of 10 XYNB, 2ml of 500 XYNB and 40ml of 50% glucose, which were mixed and then quickly poured into a sterilized petri dish.

YPD solid Medium: weighing 20g of peptone, 10g of yeast extract and 15g of agar powder, dissolving in distilled water, fixing the volume to 960ml, sterilizing at 121 ℃ for 20min under high pressure, cooling to about 60 ℃, adding 40ml of 50% glucose, mixing uniformly, and quickly pouring into a sterilized culture dish.

BMGY/BMMY medium: peptone 20g, yeast extract 10g, dissolved in 780ml distilled water, autoclaved at 121 ℃ for 20min, cooled to room temperature, and added with 100ml of 1M potassium phosphate buffer (pH6.0), 100ml of 10 XYNB, 2ml of 500 XYbiotin, and 20ml of 50% glycerol. In the case of BMMY medium, methanol is used instead of glycerol.

G418(Geneticin ) is an aminoglycoside antibiotic, purchased from Shanghai Michelin Biotechnology Ltd.

The mice used in the examples below were ICR mice purchased from experimental animal technology ltd, vindolicha, beijing.

The experimental reagents which are not particularly described in the invention are all conventional reagents in the field, and can be prepared according to the conventional method in the field or purchased from related reagent suppliers; the experimental methods not specifically described are all routine in the art, and reference may be made to relevant experimental manuals, such as molecular cloning experimental manuals or instructions of the relevant reagent manufacturers.

Example 1 design and expression of Water-soluble collagen

1. Protein design

The inventors of the present invention designed a water-soluble collagen protein comprising an α 1 chain (represented by Col1 α 1) and an α 2 chain (represented by Col1 α 2). The amino acid sequence of Col1 alpha 1 is shown in SEQ ID NO. 1, and the total length of the Col1 alpha 1 is 1422 amino acids. The amino acid sequence of Col1 alpha 2 is shown in SEQ ID NO. 2, and the total length is 1341 amino acids. The results of hydrophobicity analysis of the amino acid sequences of Col1 α 1 and Col1 α 2 by DNAMAN software are shown in fig. 1 and 2, and Col1 α 1 and Col1 α 2 have a hydrophobicity evaluation score higher than zero for only a small number of amino acids, and have good hydrophilicity.

The amino acid sequence (1422aa) of the water-soluble collagen α 1 chain (Col1 α 1) is as follows:

QEEGQEEGQEEDTPATTCTQDGQRYHDRATWKPEPCRTCTCDNGNTQCDDTTCEDTKNCPGASTPKDECCPTCPEGQTSPTDDQTTGTEGPKGDTGPRGPRGPAGPPGRDGTPGQPGQPGPPGPPGPPGPPGQGGNTAPQQSYGYDEKSAGTSTPGPMGPSGPRGQPGPPGAPGPQGTQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGQPGTAGQPGMKGHRGTSGQDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGQPGERGRPGAPGPAGARGNDGATGAAGPPGPTGPAGPPGTPGATGAKGEAGPQGARGSEGPQGTRGEPGPPGPAGAAGPAGNPGADGQPGAKGANGAPGTAGAPGTPGARGPSGPQGPSGPPGPKGNSGEPGAPGNKGDTGAKGEPGPTGTQGPPGPAGEEGKRGARGEPGPTGQPGPPGERGGPGARGTPGADGTAGPKGPAGERGAPGPAGPKGSPGEAGRPGEAGQPGAKGQTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGTMGTPGPKGAAGEPGKAGERGTPGPPGATGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGTQGQPGPAGPPGESGKPGEQGTPGDQGAPGPSGARGERGTPGERGTQGPPGPAGPRGSNGAPGNDGAKGDAGAPGAPGSQGAPGQQGMPGERGAAGQPGPKGDRGDAGPKGADGSPGKDGTRGQTGPTGPPGPAGAPGDKGETGPSGPAGPTGARGAPGDRGEPGPPGPAGTAGPPGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPTGSTGAPGPKGARGSAGPPGATGTPGAAGRTGPPGPSGNAGPPGPPGPTGKEGGKGPRGETGPAGRPGEAGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGTAGQRGTTGQPGQRGERGTPGQPGPSGEPGKQGPSGASGERGPPGPTGPPGQAGPPGESGREGSPGAEGSPGRDGSPGPKGDRGETGPAGPPGAPGAPGAPGPTGPAGKSGDRGEAGPAGPAGPTGPTGARGPAGPQGPRGDKGETGEQGDRGTKGHRGTSGQQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGQNGQPGPTGPPGPRGRTGDAGPTGPPGPPGPPGPPGPPSAGTDTSTQPQPPQEKSHDGGRYYRADDANTTRDRDQETDTTQKSQSQQTENTRSPEGSRKNPARTCRDQKMCHSDWKSGEYWTDPNQGCNQDATKTTCNMETGETCTYPTQPQTAQKNWYTSKNPKDKRHTWYGESMTDGTQTEYGGQGSDPADTATQQTTQRQMSTEASQNTTYHCKNSTAYMDQQTGNQKKAQQQQGSNETETRAEGNSRTTYSTTYDGCTSHTGAWGKTTTEYKTTKTSRQPTTDTAPQ(SEQ ID NO:1)。

the amino acid sequence (1341aa) of the water-soluble collagen alpha 2 chain (Col1 alpha 2) is as follows:

QSQQEATAGKGPTGDRGPRGERGPPGPPGRDGDDGTPGPPGPPGPPGPPGQGGNYAAQYDAKGGGPGPMGQMGPRGPPGAGAPGPQGYQGPAGEPGEPGQTGPAGARGPPGPPGKAGEDGHPGKPGRPGERGTTGPQGARGYPGTPGQPGYKGTRGHKGQDGQKGQPGAPGTKGEPGAPGENGTPGQAGARGQPGERGRTGAPGPAGARGSDGSTGPTGPAGPTGSAGPPGYPGAPGPKGEQGPTGNPGPAGPAGPRGETGQPGQSGPTGPPGNPGANGQTGAKGAAGQPGTAGAPGQPGPRGTPGPAGAAGATGARGQTGEPGPAGSKGESGNKGEPGAAGPQGPPGPSGEEGKRGPNGEPGSTGPAGPPGQRGSPGSRGQPGADGRAGTMGPAGSRGATGPAGTRGPNGDSGRPGEPGQMGPRGYPGSPGNTGPAGKEGPTGQPGTDGRPGPTGPAGARGEPGNTGYPGPKGPTGEPGKPGDKGHAGQAGARGAPGPDGNNGAQGPPGPQGTQGGKGEQGPAGPPGYQGQPGPAGTAGETGKPGERGQPGEYGQPGPAGARGERGPPGESGAAGPAGPTGSRGPSGPPGPDGNKGEPGTQGAPGTAGPSGPSGQPGERGAAGTPGGKGEKGETGQRGETGNPGRDGARGAPGATGAPGPAGANGDRGEAGAAGPAGPAGPRGSPGERGETGPAGPNGYAGPAGAAGQPGAKGERGTKGPKGENGPTGPTGPTGAAGPSGPNGPPGPAGSRGDGGPPGTTGYPGAAGRTGPPGPSGTSGPPGPPGAAGKEGQRGPRGDQGPTGRAGETGASGPPGYAGEKGPSGEPGTAGPPGTPGPQGQQGAPGTQGQPGSRGERGQPGTAGSQGEPGPQGTAGPPGARGPPGATGAPGTNGAPGEAGRDGNPGSDGPPGRDGQPGHKGERGYPGNAGPTGATGAPGPHGPTGPTGKHGNRGEPGPTGSTGPTGATGPRGPSGPQGTRGDKGEPGDKGPRGQPGTKGHNGQQGQPGQAGQHGDQGAPGSTGPAGPRGPAGPTGPTGKDGRSGQPGTTGPAGTRGSQGSQGPAGPPGPPGPPGPPGPSGGGYDYGYDGDYYRADQPRSPPSQRPKDYETDATQKSQNNQTETQQTPEGSRKNPARTCRDQRQSHPEWSSGYYWTDPNQGCTMDATKTYCDYSTGETCTRAQPENTPAKNWYRSSKAKKHTWQGETTNGGTQYEYNTEGTTTKEMATQQAYMRQQANHASQNTTYHCKNSTAYQDEETGNQKKATTQQGSNDTEQTAEGNSRYTYTTQTDGCSRKTNEWGKTTTEYKTNKPSRQPTQDTAQQDTGGADQEYGQDTGPTCYK(SEQ ID NO:2)。

2. expression vector construction

The amino acid sequences of Col1 α 1 and Col1 α 2 were translated into the corresponding DNA sequences, respectively. The sequence of the encoding gene of Col1 alpha 1 is shown in SEQ ID NO. 3, and the total length of the gene is 4266 basic groups. The sequence of the encoding gene of Col1 alpha 2 is shown in SEQ ID NO. 4, and the full length thereof is 4023 basic groups. The 5 'end of the Col1 alpha 1 coding gene sequence is added with a recognition sequence (TACGTA) of restriction enzyme SnaB I, and the 3' end is added with a recognition sequence (CCTAGG) of restriction enzyme Avr II to obtain a Col1 alpha 1 target gene sequence. The 5 'end of the encoding gene sequence of Col1 alpha 2 is added with a recognition sequence (TACGTA) of restriction enzyme SnaB I, and the 3' end is added with a recognition sequence (CCTAGG) of restriction enzyme Avr II to obtain the target gene sequence of Col1 alpha 2. The two target gene sequences are respectively subjected to whole gene synthesis by the Token biological engineering (Shanghai) corporation.

The pPIC9K plasmid was used as the expression vector. Using the restriction enzyme SnThe plasmid pPIC9K and the synthesized target gene were subjected to double digestion by aB I (Thermo Fisher Scientific) and Avr II (Thermo Fisher Scientific), respectively. Enzyme digestion system (20 μ l): ddH ₂ O16. mu.l, 10 XBuffer 2. mu.l, DNA 1. mu.l, SnaB I0.5. mu.l, Avr II 0.5. mu.l. Enzyme cutting conditions are as follows: the enzyme was cleaved at 37 ℃ for 3 h. Inactivating at 80 deg.C for 20 min.

And respectively recovering the pPIC9K plasmid and the target gene after enzyme digestion. The gene of interest was ligated into the pPIC9K vector by ligation using T4 DNA ligase (New England Biolabs). Ligation system (20 μ l): 10 Xbuffer 2. mu.l, T4 DNA ligase 0.2. mu.l, pPIC9K 3. mu.l, target gene 1. mu.l, ddH ₂ O is added to 20 μ l. Connection conditions are as follows: ligation was carried out overnight at 16 ℃.

The ligation products were transformed into E.coli strain DH5 alpha clone using heat shock method. The transformation method comprises the following steps: coli DH 5. alpha. competent cells were removed from a-80 ℃ freezer and ice-cooled for 5 min. After the glycerol of the competent cells is melted, the competent cells are added into the ligation product, the pipette tip is sucked and placed for 3-4 times, and the mixture is uniformly mixed and kept stand for 30min in ice bath. The water content of the tube wall was quickly wiped off with absorbent paper, heat-shocked at 42 ℃ for 90s, and immediately ice-bathed for 2 min. Add 800. mu.l LB liquid medium under aseptic conditions and incubate at 37 ℃ and 150rpm for 45 min. The cells were collected by centrifugation at 8000rpm for 5min, a part of the supernatant was discarded, and the remaining 100. mu.l or so of the supernatant was used to resuspend E.coli, which was then spread evenly on LB solid medium containing 100. mu.g/ml ampicillin, placed in a 37 ℃ incubator, and subjected to inverted culture for 10-16 hours. Selecting a single clone, inoculating the single clone into a liquid LB culture medium containing 100 mu g/ml ampicillin, culturing at 37 ℃ for 10-16h, sending a bacterial liquid to a biological engineering (Shanghai) corporation for sequencing, and identifying positive clones.

Plasmids of positive clones with correct sequences were extracted using a plasmid extraction kit (Omega, D6943-01) according to the kit instructions to give recombinant plasmids pPIC9K-Col1 α 1 and pPIC9K-Col1 α 2.

3. Pichia pastoris transformation

(1) Mu.g of the recombinant plasmid (pPIC9K-Col 1. alpha.1/pPIC 9K-Col 1. alpha.2) was cut by digestion with the restriction enzyme Sac I (Promega). Enzyme digestion system (20 μ l): ddH ₂ O 16μl, 1. mu.l of recombinant plasmid, 2. mu.l of 10 XBuffer, 1. mu.l of Sac I. Enzyme cutting conditions are as follows: the digestion was carried out at 37 ℃ for 2 h. After inactivation at 65 ℃ for 20min, linearized recombinant plasmids were recovered using a plasmid extraction kit (Omega, D6943-01) according to the kit instructions.

(2) And (3) taking 15 mu l of recovered linearized recombinant plasmid, uniformly mixing the linearized recombinant plasmid and 100 mu l of pichia pastoris GS115 competent cells in a 1.5ml EP tube, transferring the mixture into a 0.2cm electric shock transformation cup, standing on ice for 10min, and placing the electric shock transformation cup into an electroporator for electric shock transformation. The electric shock conditions are as follows: voltage 1.5kv, capacitance 25 muF, resistance 200 omega, shock time 10 ms.

(3) And (4) taking out the electric shock conversion cup after electric shock is finished, adding 1ml of sorbitol solution with the concentration of 1M precooled on ice into the electric shock conversion cup, and lightly blowing and uniformly mixing by using a gun head.

(4) The liquid in the electric shock conversion cup was transferred to a 2ml EP tube and shaken on a shaker at 30 ℃ for 40 min. The whole liquid is spread on MD solid medium, and cultivated at constant temperature of 30 ℃ for 2 d.

(5) And (3) picking a single colony growing on the MD solid culture medium by using an inoculating loop, inoculating the single colony to a YPD solid culture medium containing 4.0mg/ml G418, carrying out overnight culture at 30 ℃, and screening positive clones to obtain recombinant Pichia pastoris Pichia-pPIC9K-Col1 alpha 1 and Pichia-pPIC9K-Col1 alpha 2.

4. Protein expression and purification

(1) Single colonies of recombinant Pichia pastoris (Pichia-pPIC9K-Col 1. alpha.1/Pichia-pPIC 9K-Col 1. alpha.2) were picked, inoculated into Erlenmeyer flasks containing 50ml of BMGY medium, and then placed in a shaker at 30 ℃ and 220rpm overnight until OD600 ═ 1-2.

(2) Transferring the bacteria liquid into a centrifugal tube, centrifuging at 3000g for 5min at room temperature, removing supernatant, and collecting cells.

(3) Cells were resuspended at OD600 of 0.2-0.6 using BMMY broth. The culture broth was transferred to a 500ml Erlenmeyer flask, induced to express at 30 ℃ at 200rpm, and methanol was added to the medium every 24 hours to a final concentration of 1% (v/v).

(4) After the induction expression was completed, the cells were centrifuged at 3000g for 20min at 4 ℃ and the supernatant was collected for protein purification.

(5) Trichloroacetic acid (TCA) with a concentration of 100% equivalent to the volume of the supernatant of 1/9 was added to the centrifuge tube in which the supernatant was placed, mixed well with shaking, and precipitated at 4 ℃ overnight.

(6) The mixture was centrifuged at 12000rpm for 10min, the supernatant was discarded, and the precipitate was collected. And (3) inversely placing the EP pipe on the absorbent paper, and standing in a 37 ℃ oven for 10-20 min to ensure that no obvious liquid is left on the pipe wall.

(7) Adding 200 μ l of cold propanol, shaking, mixing, standing at room temperature for 10min, and washing off residual TCA on tube wall and tube bottom.

(8) And (5) repeating the steps (6) and (7) for 2-3 times to obtain purified proteins Col1 alpha 1 and Col1 alpha 2.

5. SDS-PAGE detection of proteins

Preparing SDS-PAGE protein electrophoresis gel, wherein the concentration of separation gel is 10 percent, the concentration of concentrated gel is 5 percent, and the formula is as follows:

10% of separation gel: 3.3ml of 30% acrylamide solution, ddH ₂ O4 ml, gel buffer 2.5ml, 10% SDS 0.1ml, 10% AP solution 0.1ml, TEMED 0.004 ml.

5% of concentrated gel: 0.67ml of 30% acrylamide solution, ddH ₂ O2.7 ml, glue buffer 0.5ml, 10% SDS 0.04ml, 10% AP solution 0.04ml, TEMED 0.04 ml.

Sample treatment: adding a proper amount of protein to be detected into a Loading Buffer, shaking and uniformly mixing, boiling in boiling water for 5-10 mim to denature the protein, centrifuging at 12000rpm for 10min, and taking supernatant. Load 10. mu.l. Electrophoresis parameters: constant pressure is 80V, and the pressure is 120V after the separation gel is fed.

The SDS-PAGE results are shown in FIG. 3, and the molecular weight of the protein of Col1 alpha 1 is about 130kDa, and the molecular weight of the protein of Col1 alpha 2 is about 125kDa, which is consistent with the expected molecular weight of the protein.

Example 2 anti-fatigue test in mice

1. Weight bearing swimming experiment

40 healthy male mice with the weight of 18-22 g are selected. Mice were randomly divided into 4 groups, including 1 blank group and 3 experimental groups, with 10 mice per group.

Each mouse in the blank group was gavaged with 1g of physiological saline (aqueous sodium chloride solution having a concentration of 0.9% g/ml) daily. Each mouse of the experimental group was administered daily the purified protein of example 1, specifically: mixing Col1 alpha 1 and Col1 alpha 2 at a molar ratio of 2:1, mixing 0.1g of the mixture with 1g of normal saline, and performing intragastric administration. The drug is continuously administered for 7 days, and after 2 hours of the last intragastric gavage, the mice are placed in a swimming box for a load swimming experiment.

The load swimming test is an animal model for evaluating the anti-fatigue effect of a substance. The swimming box of the experiment has the water depth of 30cm, the water temperature (22 +/-1) DEG C, and the tail root of the rat is loaded with 10 percent of weight of lead skin. The time from the start of swimming until the head of the mouse fully sinks into the water and the mouse cannot float out of the water surface for 8 seconds (in a depleted state) was recorded as the mouse weight swimming time. This weight swimming time is regarded as an index for quantitatively and objectively evaluating the degree of fatigue. The longer the weight swimming time, the better the anti-fatigue effect of the protein.

2. Pole climbing experiment

Each mouse in the blank group was gavaged with 1g of physiological saline daily. Each mouse of the experimental group was administered daily the purified protein of example 1, specifically: mixing Col1 alpha 1 and Col1 alpha 2 at a molar ratio of 2:1, mixing 0.1g of the mixture with 1g of normal saline, and performing intragastric administration. The drug is continuously administrated for 7 days, and a climbing rod experiment is carried out after the last intragastric gavage for 2 hours.

The equipment for climbing rod experiment is climbing rod frame. The pole-climbing frame of this experiment is the organic glass round bar that 120 mesh abrasive paper polished of diameter 10mm, length 50cm, and its upper end is fixed in on the plank, and the lower extreme is apart from ground 20 cm. The method of the climbing rod experiment is as follows: the mouse is placed at the top end of the organic glass round bar of the climbing rod frame, so that the muscle of the mouse is in a static tension state, and the time of the mouse falling from the organic glass round bar due to muscle fatigue is recorded. The experiment was stopped at the 3 rd fall, and the time of 3 cumulative times was taken as the pole climbing time of the mouse.

3. Determination of biochemical index

60 healthy male mice with the weight of 18-22 g are selected. Mice were randomly divided into 6 groups, including 3 blank groups and 3 experimental groups, with 10 mice per group.

Each mouse in the blank group was gavaged with 1g of physiological saline daily. Each mouse of the experimental group was administered daily the purified protein of example 1, specifically: mixing Col1 alpha 1 and Col1 alpha 2 at a molar ratio of 2:1, mixing 0.1g of the mixture with 1g of normal saline, intragastrically administering, and continuously administering for 7 d. The following experiment was performed 0.5h after the last administration.

Randomly selecting a blank group and an experimental group, placing in a water tank with the water temperature of 22 +/-1 ℃ for swimming for 90min, collecting eyeball blood after swimming for 60min, placing at 4 ℃ for 3h for separating serum, and measuring the urea nitrogen in the serum by using a urea nitrogen measuring kit (G-GLONE gold clone, SH960K-50) according to the kit specification.

Randomly selecting a blank group and an experimental group, placing the blank group and the experimental group in a water tank with the water temperature of 22 +/-1 ℃ for swimming for 30min, collecting eyeball blood after the swimming is finished, collecting the eyeball blood again after the swimming is quiet for 30min, and respectively measuring the blood lactic acid concentration of the mouse before the swimming, just after the swimming (0 min after the swimming) and quiet for 30min after the swimming by using a lactic acid LAC detection kit (Solarbio Soilebao, BC2230-50T/24S) according to the kit specification.

A blank group and an experimental group were randomly selected, 50mg of liver was weighed for each mouse, and liver glycogen was measured using the anthrone method. The procedures of the anthrone method are described in "Gaoshan, Tong-ying, Xiong-Yan-si, et al, comparative study of the anthrone method and the kit method for determination of glycogen content, first public health, 2022,5(1): 38-40".

4. Results of the experiment

The results of the experiment are shown in tables 1 and 2, and fig. 4 and 5. The weight swimming time and pole climbing time for each group in table 1 are the mean ± standard deviation of the weight swimming time and pole climbing time for each group of 10 mice. The biochemical indicators of the mice in Table 2 are the average. + -. standard deviation of the biochemical indicator measurement results of 10 mice in each group.

The data show that compared with the blank group, the experimental group prolongs the weight-bearing swimming time of the mice by about 50%, the pole-climbing time of the mice by nearly 1 time, the urea nitrogen level and the lactic acid level in the bodies of the mice are reduced, and the liver glycogen level is improved, which shows that the water-soluble collagen is effectively absorbed and utilized by the organisms, and the anti-fatigue capability of the animals is obviously improved.

TABLE 1 mouse weight bearing swimming experiment and pole climbing experiment results

Group of	Number of animals/animal	Weight swimming time/min	Pole climbing time/min
				Blank group
	10	46.4±18.8	4.3±2.2
				Experimental group 1	10	66.5±25.1	7.4±2.5
Experimental group 2	10	72.6±11.3	8.4±2.1
				Experimental group 3	10	65.8±12.8	7.3±2.9

TABLE 2 Biochemical index measurement results of mice

SEQUENCE LISTING

<110> university of Chongqing

<120> water-soluble collagen and application thereof

<130> P2230427-CQD-CQ-TXH

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1422

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of alpha 1 chain of water-soluble collagen

<400> 1

Gln Glu Glu Gly Gln Glu Glu Gly Gln Glu Glu Asp Thr Pro Ala Thr

1 5 10 15

Thr Cys Thr Gln Asp Gly Gln Arg Tyr His Asp Arg Ala Thr Trp Lys

20 25 30

Pro Glu Pro Cys Arg Thr Cys Thr Cys Asp Asn Gly Asn Thr Gln Cys

35 40 45

Asp Asp Thr Thr Cys Glu Asp Thr Lys Asn Cys Pro Gly Ala Ser Thr

50 55 60

Pro Lys Asp Glu Cys Cys Pro Thr Cys Pro Glu Gly Gln Thr Ser Pro

65 70 75 80

Thr Asp Asp Gln Thr Thr Gly Thr Glu Gly Pro Lys Gly Asp Thr Gly

85 90 95

Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro Pro Gly Arg Asp Gly Thr

100 105 110

Pro Gly Gln Pro Gly Gln Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro

115 120 125

Gly Pro Pro Gly Gln Gly Gly Asn Thr Ala Pro Gln Gln Ser Tyr Gly

130 135 140

Tyr Asp Glu Lys Ser Ala Gly Thr Ser Thr Pro Gly Pro Met Gly Pro

145 150 155 160

Ser Gly Pro Arg Gly Gln Pro Gly Pro Pro Gly Ala Pro Gly Pro Gln

165 170 175

Gly Thr Gln Gly Pro Pro Gly Glu Pro Gly Glu Pro Gly Ala Ser Gly

180 185 190

Pro Met Gly Pro Arg Gly Pro Pro Gly Pro Pro Gly Lys Asn Gly Asp

195 200 205

Asp Gly Glu Ala Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Pro Pro

210 215 220

Gly Pro Gln Gly Ala Arg Gly Gln Pro Gly Thr Ala Gly Gln Pro Gly

225 230 235 240

Met Lys Gly His Arg Gly Thr Ser Gly Gln Asp Gly Ala Lys Gly Asp

245 250 255

Ala Gly Pro Ala Gly Pro Lys Gly Glu Pro Gly Ser Pro Gly Glu Asn

260 265 270

Gly Ala Pro Gly Gln Met Gly Pro Arg Gly Gln Pro Gly Glu Arg Gly

275 280 285

Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Asn Asp Gly Ala

290 295 300

Thr Gly Ala Ala Gly Pro Pro Gly Pro Thr Gly Pro Ala Gly Pro Pro

305 310 315 320

Gly Thr Pro Gly Ala Thr Gly Ala Lys Gly Glu Ala Gly Pro Gln Gly

325 330 335

Ala Arg Gly Ser Glu Gly Pro Gln Gly Thr Arg Gly Glu Pro Gly Pro

340 345 350

Pro Gly Pro Ala Gly Ala Ala Gly Pro Ala Gly Asn Pro Gly Ala Asp

355 360 365

Gly Gln Pro Gly Ala Lys Gly Ala Asn Gly Ala Pro Gly Thr Ala Gly

370 375 380

Ala Pro Gly Thr Pro Gly Ala Arg Gly Pro Ser Gly Pro Gln Gly Pro

385 390 395 400

Ser Gly Pro Pro Gly Pro Lys Gly Asn Ser Gly Glu Pro Gly Ala Pro

405 410 415

Gly Asn Lys Gly Asp Thr Gly Ala Lys Gly Glu Pro Gly Pro Thr Gly

420 425 430

Thr Gln Gly Pro Pro Gly Pro Ala Gly Glu Glu Gly Lys Arg Gly Ala

435 440 445

Arg Gly Glu Pro Gly Pro Thr Gly Gln Pro Gly Pro Pro Gly Glu Arg

450 455 460

Gly Gly Pro Gly Ala Arg Gly Thr Pro Gly Ala Asp Gly Thr Ala Gly

465 470 475 480

Pro Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro Gly Pro Ala Gly Pro

485 490 495

Lys Gly Ser Pro Gly Glu Ala Gly Arg Pro Gly Glu Ala Gly Gln Pro

500 505 510

Gly Ala Lys Gly Gln Thr Gly Ser Pro Gly Ser Pro Gly Pro Asp Gly

515 520 525

Lys Thr Gly Pro Pro Gly Pro Ala Gly Gln Asp Gly Arg Pro Gly Pro

530 535 540

Pro Gly Pro Pro Gly Ala Arg Gly Gln Ala Gly Thr Met Gly Thr Pro

545 550 555 560

Gly Pro Lys Gly Ala Ala Gly Glu Pro Gly Lys Ala Gly Glu Arg Gly

565 570 575

Thr Pro Gly Pro Pro Gly Ala Thr Gly Pro Ala Gly Lys Asp Gly Glu

580 585 590

Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu Arg

595 600 605

Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly Thr Gln Gly Gln Pro Gly

610 615 620

Pro Ala Gly Pro Pro Gly Glu Ser Gly Lys Pro Gly Glu Gln Gly Thr

625 630 635 640

Pro Gly Asp Gln Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu Arg

645 650 655

Gly Thr Pro Gly Glu Arg Gly Thr Gln Gly Pro Pro Gly Pro Ala Gly

660 665 670

Pro Arg Gly Ser Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly Asp

675 680 685

Ala Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Gln Gln

690 695 700

Gly Met Pro Gly Glu Arg Gly Ala Ala Gly Gln Pro Gly Pro Lys Gly

705 710 715 720

Asp Arg Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Lys

725 730 735

Asp Gly Thr Arg Gly Gln Thr Gly Pro Thr Gly Pro Pro Gly Pro Ala

740 745 750

Gly Ala Pro Gly Asp Lys Gly Glu Thr Gly Pro Ser Gly Pro Ala Gly

755 760 765

Pro Thr Gly Ala Arg Gly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro

770 775 780

Pro Gly Pro Ala Gly Thr Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro

785 790 795 800

Gly Ala Lys Gly Glu Pro Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly

805 810 815

Pro Pro Gly Pro Ala Gly Pro Ala Gly Pro Pro Gly Pro Thr Gly Ser

820 825 830

Thr Gly Ala Pro Gly Pro Lys Gly Ala Arg Gly Ser Ala Gly Pro Pro

835 840 845

Gly Ala Thr Gly Thr Pro Gly Ala Ala Gly Arg Thr Gly Pro Pro Gly

850 855 860

Pro Ser Gly Asn Ala Gly Pro Pro Gly Pro Pro Gly Pro Thr Gly Lys

865 870 875 880

Glu Gly Gly Lys Gly Pro Arg Gly Glu Thr Gly Pro Ala Gly Arg Pro

885 890 895

Gly Glu Ala Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Glu Lys Gly

900 905 910

Ser Pro Gly Ala Asp Gly Pro Ala Gly Ala Pro Gly Thr Pro Gly Pro

915 920 925

Gln Gly Thr Ala Gly Gln Arg Gly Thr Thr Gly Gln Pro Gly Gln Arg

930 935 940

Gly Glu Arg Gly Thr Pro Gly Gln Pro Gly Pro Ser Gly Glu Pro Gly

945 950 955 960

Lys Gln Gly Pro Ser Gly Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro

965 970 975

Thr Gly Pro Pro Gly Gln Ala Gly Pro Pro Gly Glu Ser Gly Arg Glu

980 985 990

Gly Ser Pro Gly Ala Glu Gly Ser Pro Gly Arg Asp Gly Ser Pro Gly

995 1000 1005

Pro Lys Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro Pro Gly

1010 1015 1020

Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro Thr Gly Pro Ala Gly

1025 1030 1035

Lys Ser Gly Asp Arg Gly Glu Ala Gly Pro Ala Gly Pro Ala Gly

1040 1045 1050

Pro Thr Gly Pro Thr Gly Ala Arg Gly Pro Ala Gly Pro Gln Gly

1055 1060 1065

Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp Arg Gly

1070 1075 1080

Thr Lys Gly His Arg Gly Thr Ser Gly Gln Gln Gly Pro Pro Gly

1085 1090 1095

Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly

1100 1105 1110

Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly

1115 1120 1125

Lys Asp Gly Gln Asn Gly Gln Pro Gly Pro Thr Gly Pro Pro Gly

1130 1135 1140

Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Thr Gly Pro Pro Gly

1145 1150 1155

Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Thr

1160 1165 1170

Asp Thr Ser Thr Gln Pro Gln Pro Pro Gln Glu Lys Ser His Asp

1175 1180 1185

Gly Gly Arg Tyr Tyr Arg Ala Asp Asp Ala Asn Thr Thr Arg Asp

1190 1195 1200

Arg Asp Gln Glu Thr Asp Thr Thr Gln Lys Ser Gln Ser Gln Gln

1205 1210 1215

Thr Glu Asn Thr Arg Ser Pro Glu Gly Ser Arg Lys Asn Pro Ala

1220 1225 1230

Arg Thr Cys Arg Asp Gln Lys Met Cys His Ser Asp Trp Lys Ser

1235 1240 1245

Gly Glu Tyr Trp Thr Asp Pro Asn Gln Gly Cys Asn Gln Asp Ala

1250 1255 1260

Thr Lys Thr Thr Cys Asn Met Glu Thr Gly Glu Thr Cys Thr Tyr

1265 1270 1275

Pro Thr Gln Pro Gln Thr Ala Gln Lys Asn Trp Tyr Thr Ser Lys

1280 1285 1290

Asn Pro Lys Asp Lys Arg His Thr Trp Tyr Gly Glu Ser Met Thr

1295 1300 1305

Asp Gly Thr Gln Thr Glu Tyr Gly Gly Gln Gly Ser Asp Pro Ala

1310 1315 1320

Asp Thr Ala Thr Gln Gln Thr Thr Gln Arg Gln Met Ser Thr Glu

1325 1330 1335

Ala Ser Gln Asn Thr Thr Tyr His Cys Lys Asn Ser Thr Ala Tyr

1340 1345 1350

Met Asp Gln Gln Thr Gly Asn Gln Lys Lys Ala Gln Gln Gln Gln

1355 1360 1365

Gly Ser Asn Glu Thr Glu Thr Arg Ala Glu Gly Asn Ser Arg Thr

1370 1375 1380

Thr Tyr Ser Thr Thr Tyr Asp Gly Cys Thr Ser His Thr Gly Ala

1385 1390 1395

Trp Gly Lys Thr Thr Thr Glu Tyr Lys Thr Thr Lys Thr Ser Arg

1400 1405 1410

Gln Pro Thr Thr Asp Thr Ala Pro Gln

1415 1420

<210> 2

<211> 1341

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of alpha 2 chain of water-soluble collagen

<400> 2

Gln Ser Gln Gln Glu Ala Thr Ala Gly Lys Gly Pro Thr Gly Asp Arg

1 5 10 15

Gly Pro Arg Gly Glu Arg Gly Pro Pro Gly Pro Pro Gly Arg Asp Gly

20 25 30

Asp Asp Gly Thr Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro

35 40 45

Pro Gly Gln Gly Gly Asn Tyr Ala Ala Gln Tyr Asp Ala Lys Gly Gly

50 55 60

Gly Pro Gly Pro Met Gly Gln Met Gly Pro Arg Gly Pro Pro Gly Ala

65 70 75 80

Gly Ala Pro Gly Pro Gln Gly Tyr Gln Gly Pro Ala Gly Glu Pro Gly

85 90 95

Glu Pro Gly Gln Thr Gly Pro Ala Gly Ala Arg Gly Pro Pro Gly Pro

100 105 110

Pro Gly Lys Ala Gly Glu Asp Gly His Pro Gly Lys Pro Gly Arg Pro

115 120 125

Gly Glu Arg Gly Thr Thr Gly Pro Gln Gly Ala Arg Gly Tyr Pro Gly

130 135 140

Thr Pro Gly Gln Pro Gly Tyr Lys Gly Thr Arg Gly His Lys Gly Gln

145 150 155 160

Asp Gly Gln Lys Gly Gln Pro Gly Ala Pro Gly Thr Lys Gly Glu Pro

165 170 175

Gly Ala Pro Gly Glu Asn Gly Thr Pro Gly Gln Ala Gly Ala Arg Gly

180 185 190

Gln Pro Gly Glu Arg Gly Arg Thr Gly Ala Pro Gly Pro Ala Gly Ala

195 200 205

Arg Gly Ser Asp Gly Ser Thr Gly Pro Thr Gly Pro Ala Gly Pro Thr

210 215 220

Gly Ser Ala Gly Pro Pro Gly Tyr Pro Gly Ala Pro Gly Pro Lys Gly

225 230 235 240

Glu Gln Gly Pro Thr Gly Asn Pro Gly Pro Ala Gly Pro Ala Gly Pro

245 250 255

Arg Gly Glu Thr Gly Gln Pro Gly Gln Ser Gly Pro Thr Gly Pro Pro

260 265 270

Gly Asn Pro Gly Ala Asn Gly Gln Thr Gly Ala Lys Gly Ala Ala Gly

275 280 285

Gln Pro Gly Thr Ala Gly Ala Pro Gly Gln Pro Gly Pro Arg Gly Thr

290 295 300

Pro Gly Pro Ala Gly Ala Ala Gly Ala Thr Gly Ala Arg Gly Gln Thr

305 310 315 320

Gly Glu Pro Gly Pro Ala Gly Ser Lys Gly Glu Ser Gly Asn Lys Gly

325 330 335

Glu Pro Gly Ala Ala Gly Pro Gln Gly Pro Pro Gly Pro Ser Gly Glu

340 345 350

Glu Gly Lys Arg Gly Pro Asn Gly Glu Pro Gly Ser Thr Gly Pro Ala

355 360 365

Gly Pro Pro Gly Gln Arg Gly Ser Pro Gly Ser Arg Gly Gln Pro Gly

370 375 380

Ala Asp Gly Arg Ala Gly Thr Met Gly Pro Ala Gly Ser Arg Gly Ala

385 390 395 400

Thr Gly Pro Ala Gly Thr Arg Gly Pro Asn Gly Asp Ser Gly Arg Pro

405 410 415

Gly Glu Pro Gly Gln Met Gly Pro Arg Gly Tyr Pro Gly Ser Pro Gly

420 425 430

Asn Thr Gly Pro Ala Gly Lys Glu Gly Pro Thr Gly Gln Pro Gly Thr

435 440 445

Asp Gly Arg Pro Gly Pro Thr Gly Pro Ala Gly Ala Arg Gly Glu Pro

450 455 460

Gly Asn Thr Gly Tyr Pro Gly Pro Lys Gly Pro Thr Gly Glu Pro Gly

465 470 475 480

Lys Pro Gly Asp Lys Gly His Ala Gly Gln Ala Gly Ala Arg Gly Ala

485 490 495

Pro Gly Pro Asp Gly Asn Asn Gly Ala Gln Gly Pro Pro Gly Pro Gln

500 505 510

Gly Thr Gln Gly Gly Lys Gly Glu Gln Gly Pro Ala Gly Pro Pro Gly

515 520 525

Tyr Gln Gly Gln Pro Gly Pro Ala Gly Thr Ala Gly Glu Thr Gly Lys

530 535 540

Pro Gly Glu Arg Gly Gln Pro Gly Glu Tyr Gly Gln Pro Gly Pro Ala

545 550 555 560

Gly Ala Arg Gly Glu Arg Gly Pro Pro Gly Glu Ser Gly Ala Ala Gly

565 570 575

Pro Ala Gly Pro Thr Gly Ser Arg Gly Pro Ser Gly Pro Pro Gly Pro

580 585 590

Asp Gly Asn Lys Gly Glu Pro Gly Thr Gln Gly Ala Pro Gly Thr Ala

595 600 605

Gly Pro Ser Gly Pro Ser Gly Gln Pro Gly Glu Arg Gly Ala Ala Gly

610 615 620

Thr Pro Gly Gly Lys Gly Glu Lys Gly Glu Thr Gly Gln Arg Gly Glu

625 630 635 640

Thr Gly Asn Pro Gly Arg Asp Gly Ala Arg Gly Ala Pro Gly Ala Thr

645 650 655

Gly Ala Pro Gly Pro Ala Gly Ala Asn Gly Asp Arg Gly Glu Ala Gly

660 665 670

Ala Ala Gly Pro Ala Gly Pro Ala Gly Pro Arg Gly Ser Pro Gly Glu

675 680 685

Arg Gly Glu Thr Gly Pro Ala Gly Pro Asn Gly Tyr Ala Gly Pro Ala

690 695 700

Gly Ala Ala Gly Gln Pro Gly Ala Lys Gly Glu Arg Gly Thr Lys Gly

705 710 715 720

Pro Lys Gly Glu Asn Gly Pro Thr Gly Pro Thr Gly Pro Thr Gly Ala

725 730 735

Ala Gly Pro Ser Gly Pro Asn Gly Pro Pro Gly Pro Ala Gly Ser Arg

740 745 750

Gly Asp Gly Gly Pro Pro Gly Thr Thr Gly Tyr Pro Gly Ala Ala Gly

755 760 765

Arg Thr Gly Pro Pro Gly Pro Ser Gly Thr Ser Gly Pro Pro Gly Pro

770 775 780

Pro Gly Ala Ala Gly Lys Glu Gly Gln Arg Gly Pro Arg Gly Asp Gln

785 790 795 800

Gly Pro Thr Gly Arg Ala Gly Glu Thr Gly Ala Ser Gly Pro Pro Gly

805 810 815

Tyr Ala Gly Glu Lys Gly Pro Ser Gly Glu Pro Gly Thr Ala Gly Pro

820 825 830

Pro Gly Thr Pro Gly Pro Gln Gly Gln Gln Gly Ala Pro Gly Thr Gln

835 840 845

Gly Gln Pro Gly Ser Arg Gly Glu Arg Gly Gln Pro Gly Thr Ala Gly

850 855 860

Ser Gln Gly Glu Pro Gly Pro Gln Gly Thr Ala Gly Pro Pro Gly Ala

865 870 875 880

Arg Gly Pro Pro Gly Ala Thr Gly Ala Pro Gly Thr Asn Gly Ala Pro

885 890 895

Gly Glu Ala Gly Arg Asp Gly Asn Pro Gly Ser Asp Gly Pro Pro Gly

900 905 910

Arg Asp Gly Gln Pro Gly His Lys Gly Glu Arg Gly Tyr Pro Gly Asn

915 920 925

Ala Gly Pro Thr Gly Ala Thr Gly Ala Pro Gly Pro His Gly Pro Thr

930 935 940

Gly Pro Thr Gly Lys His Gly Asn Arg Gly Glu Pro Gly Pro Thr Gly

945 950 955 960

Ser Thr Gly Pro Thr Gly Ala Thr Gly Pro Arg Gly Pro Ser Gly Pro

965 970 975

Gln Gly Thr Arg Gly Asp Lys Gly Glu Pro Gly Asp Lys Gly Pro Arg

980 985 990

Gly Gln Pro Gly Thr Lys Gly His Asn Gly Gln Gln Gly Gln Pro Gly

995 1000 1005

Gln Ala Gly Gln His Gly Asp Gln Gly Ala Pro Gly Ser Thr Gly

1010 1015 1020

Pro Ala Gly Pro Arg Gly Pro Ala Gly Pro Thr Gly Pro Thr Gly

1025 1030 1035

Lys Asp Gly Arg Ser Gly Gln Pro Gly Thr Thr Gly Pro Ala Gly

1040 1045 1050

Thr Arg Gly Ser Gln Gly Ser Gln Gly Pro Ala Gly Pro Pro Gly

1055 1060 1065

Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Ser Gly Gly Gly Tyr

1070 1075 1080

Asp Tyr Gly Tyr Asp Gly Asp Tyr Tyr Arg Ala Asp Gln Pro Arg

1085 1090 1095

Ser Pro Pro Ser Gln Arg Pro Lys Asp Tyr Glu Thr Asp Ala Thr

1100 1105 1110

Gln Lys Ser Gln Asn Asn Gln Thr Glu Thr Gln Gln Thr Pro Glu

1115 1120 1125

Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Gln Arg Gln

1130 1135 1140

Ser His Pro Glu Trp Ser Ser Gly Tyr Tyr Trp Thr Asp Pro Asn

1145 1150 1155

Gln Gly Cys Thr Met Asp Ala Thr Lys Thr Tyr Cys Asp Tyr Ser

1160 1165 1170

Thr Gly Glu Thr Cys Thr Arg Ala Gln Pro Glu Asn Thr Pro Ala

1175 1180 1185

Lys Asn Trp Tyr Arg Ser Ser Lys Ala Lys Lys His Thr Trp Gln

1190 1195 1200

Gly Glu Thr Thr Asn Gly Gly Thr Gln Tyr Glu Tyr Asn Thr Glu

1205 1210 1215

Gly Thr Thr Thr Lys Glu Met Ala Thr Gln Gln Ala Tyr Met Arg

1220 1225 1230

Gln Gln Ala Asn His Ala Ser Gln Asn Thr Thr Tyr His Cys Lys

1235 1240 1245

Asn Ser Thr Ala Tyr Gln Asp Glu Glu Thr Gly Asn Gln Lys Lys

1250 1255 1260

Ala Thr Thr Gln Gln Gly Ser Asn Asp Thr Glu Gln Thr Ala Glu

1265 1270 1275

Gly Asn Ser Arg Tyr Thr Tyr Thr Thr Gln Thr Asp Gly Cys Ser

1280 1285 1290

Arg Lys Thr Asn Glu Trp Gly Lys Thr Thr Thr Glu Tyr Lys Thr

1295 1300 1305

Asn Lys Pro Ser Arg Gln Pro Thr Gln Asp Thr Ala Gln Gln Asp

1310 1315 1320

Thr Gly Gly Ala Asp Gln Glu Tyr Gly Gln Asp Thr Gly Pro Thr

1325 1330 1335

Cys Tyr Lys

1340

<210> 3

<211> 4266

<212> DNA

<213> Artificial Sequence

<220>

<223> coding gene sequence of water-soluble collagen alpha 1 chain

<400> 3

caagaggaag gtcaggaaga aggacaagag gaagacactc cagctactac atgcactcaa 60

gacggacaaa gataccatga tcgtgccacg tggaagccag aaccttgtcg aacttgcact 120

tgcgataacg gtaatactca gtgcgacgac accacctgtg aagacaccaa gaattgccct 180

ggtgctagta ctccaaaaga tgaatgttgt ccaacttgtc ctgagggtca aacttcacca 240

actgacgacc aaaccacggg taccgaagga cctaagggtg acactggtcc tagaggtcct 300

agaggacctg ctggtccacc tggtagagac ggaactcctg gtcaaccagg ccaaccagga 360

ccacctggac cacctggacc accaggacca ccaggtcaag gtggtaatac tgctcctcag 420

caatcttatg gttatgatga aaagtccgcc ggaacatcta ctccaggtcc tatgggtcca 480

tctggaccta gaggtcaacc cggtcctcct ggtgcacctg gtcctcaagg tacccaaggt 540

ccacctggtg aacctggtga acctggagct tccggtccaa tgggtcctag aggtcctcca 600

ggtccacctg gaaaaaacgg tgatgatggt gaagctggta agccaggtag accaggagaa 660

agaggacctc ccggtcccca aggtgctaga ggtcagcctg gcaccgctgg acaaccaggt 720

atgaaaggac atagaggaac ctcaggtcag gatggagcta aaggtgatgc cggtccagcc 780

ggtccaaaag gagagccagg tagtccagga gagaacggcg caccaggaca aatgggtcca 840

aggggtcagc ctggagaacg tggtagacct ggtgctccag gtcccgcagg agctagaggt 900

aacgatggtg caactggtgc tgctggtcct cctggtccta ctggtccagc tggtccacct 960

ggtacacccg gtgctactgg tgctaaaggt gaagccggtc ctcaaggtgc taggggttct 1020

gagggaccac aaggaactag aggagaacca ggacctccag gaccagctgg agctgctggt 1080

ccagctggaa atccaggtgc agacggtcaa ccaggtgcca agggtgctaa tggtgcaccc 1140

ggaactgctg gtgcacctgg taccccaggt gctagaggtc caagtggtcc acagggtcca 1200

tccggtccac ctggtcctaa aggtaattct ggagagccag gtgcacctgg aaacaaaggt 1260

gacactggag ccaaaggtga gcctggtcct actggtacgc aaggtccacc tggcccagct 1320

ggagaggaag gtaagagagg tgctagaggt gaacccggac caaccggaca acctggacct 1380

ccaggtgaac gtggaggtcc tggagccaga ggtacgcctg gtgcagacgg tactgctggt 1440

ccaaaaggac ctgccggaga aagaggtgcc cctggaccag ctggcccaaa aggttctcca 1500

ggtgaggcag gaagaccagg cgaagcaggt caaccaggcg ccaagggaca aactggaagt 1560

ccaggctccc ccggtccaga tggaaaaacc ggaccacctg gtccagctgg acaagatggt 1620

agaccaggtc ctccaggacc acctggagct agaggacaag ccggtaccat gggtactcct 1680

ggacctaaag gtgcagctgg tgagccagga aaagctggtg agagaggtac cccaggtcct 1740

ccaggagcta ctggtccagc tggtaaggac ggagaagctg gagcacaagg accaccagga 1800

cctgcaggac ctgctggtga acgtggagaa cagggaccag ctggttcacc aggtactcag 1860

ggtcaaccag gaccagccgg accacctggt gagtctggta agccaggaga acagggaacc 1920

cctggtgatc aaggtgctcc tggtccttct ggagcaagag gagaacgtgg aactcctgga 1980

gaaagaggaa ctcaaggtcc acctggacct gccggtccta gaggttcaaa tggtgcacca 2040

ggaaatgacg gagctaaggg cgatgctggt gcacctggtg ctccaggatc acaaggtgca 2100

cctggacaac aaggcatgcc aggagaaaga ggtgcagctg gtcagccagg acctaagggt 2160

gacagaggtg atgctggacc taaaggagct gacggttcac ccggtaaaga tggtactaga 2220

ggtcagactg gtcctacagg accaccagga ccagctggag cacctggtga taaaggagag 2280

actggccctt ctggacctgc tggtccaact ggtgccagag gtgccccagg agatagaggc 2340

gaaccaggtc caccaggtcc agcaggtact gctggtcctc ccggtgctga tggtcaacca 2400

ggtgctaaag gtgagccagg agacgctggt gccaaaggtg atgctggtcc tcctggtcca 2460

gcaggtccag caggaccacc tggtcctact ggttcaacag gagcccctgg accaaaaggt 2520

gcaagaggat cagctggccc acctggtgct actggtacgc caggagccgc aggaagaaca 2580

ggtcctcctg gacctagtgg taatgctgga cctcctggtc ccccaggacc tacaggaaag 2640

gagggaggca aaggaccacg aggagaaacc ggtcctgccg gtagacctgg tgaagctgga 2700

ccaccaggtc ctcctggtcc agctggagag aaaggctcac caggcgctga tggtccagca 2760

ggtgcacctg gtactcccgg tcctcagggt actgctggtc agagaggcac caccggacag 2820

cctggacaaa ggggtgagag aggtacacct ggacaacctg gtccatctgg cgaacctggt 2880

aagcaaggtc cttctggagc ctctggagaa aggggtccac ctggtccaac cggtccacct 2940

ggacaagctg gaccaccagg tgaatctggt agggagggat ctccaggtgc tgagggctcc 3000

ccaggaagag acggatctcc tggtccaaag ggtgatagag gtgagaccgg tccagctggt 3060

ccaccaggtg ctccaggagc acctggagca cctggtccaa ctggaccagc aggtaagtcc 3120

ggtgatagag gtgaagctgg acctgcagga cctgccggac ctactggtcc aacaggtgcc 3180

agaggtcccg ctggtcctca aggaccaagg ggagataaag gagagacggg tgaacaagga 3240

gatcgtggta ccaagggcca tcgtggaact tcaggacaac aaggtcctcc aggtccacct 3300

ggttctccag gtgaacaggg tccctctgga gcttcaggtc cagcaggacc aagaggccct 3360

ccaggtagtg caggtgcacc aggaaaagat ggtcaaaatg gtcaaccagg tcctaccggt 3420

ccaccaggtc caagaggtag aactggtgat gctggaccaa ccggtccacc tggacctcct 3480

ggacctccag gtcctccagg acctccttca gctggtaccg acacctcaac ccaaccacaa 3540

ccaccccaag agaagtctca cgatggtggt agatactata gagctgatga cgccaacaca 3600

actagggatc gtgaccaaga gactgacaca acacagaagt ctcagtccca gcagacagag 3660

aacactagat ctccagaggg tagtagaaaa aatcctgcaa ggacctgtag agatcaaaaa 3720

atgtgccact cagattggaa atccggtgag tactggactg atcctaacca aggttgtaac 3780

caggacgcta ctaaaacaac ttgtaacatg gaaactggtg aaacttgtac ctatcctacc 3840

caaccacaaa ctgcccaaaa aaactggtac acttccaaga accctaaaga taaaagacat 3900

acatggtacg gagaaagtat gactgatgga acacaaactg aatatggtgg acaaggatcc 3960

gatcccgcag ataccgctac tcagcaaact acacagagac aaatgtcaac cgaggccagt 4020

cagaatacaa cttatcactg taagaattct accgcctata tggaccagca gactggaaat 4080

cagaagaagg ctcaacaaca acaaggtagt aacgaaactg agacacgtgc cgagggaaac 4140

agtagaacaa cgtactctac aacttatgac ggatgtactt ctcacacggg agcatggggt 4200

aagaccacga ctgaatataa gacaacaaag acatcaagac aacccaccac cgacacagct 4260

ccacag 4266

<210> 4

<211> 4023

<212> DNA

<213> Artificial Sequence

<220>

<223> coding gene sequence of water-soluble collagen alpha 2 chain

<400> 4

cagtctcaac aggaagcaac tgctggaaag ggtcccacag gtgatagagg tccacgtggt 60

gagcgaggac ctccaggacc tccaggaaga gacggagatg acggaactcc tggtccacct 120

ggtccacctg gaccacctgg accacctggt caaggtggaa attatgcagc tcaatatgat 180

gccaagggag gtggaccagg tccaatggga caaatgggac ctagaggacc tcctggagct 240

ggagcacctg gtcctcaggg ataccaaggt cccgctggtg aaccaggcga accaggtcaa 300

acaggaccag ccggtgcaag aggtccacca ggtccaccag gtaaggctgg tgaagacgga 360

catccaggaa aaccaggaag gcctggagaa cgtggtacta ccggcccaca aggtgctcgt 420

ggatatcctg gaactcctgg acaaccagga tataagggta caagaggaca caaaggacag 480

gatggacaga agggtcaacc tggtgctcct ggaactaaag gtgaacctgg agcaccagga 540

gaaaacggaa cccctggtca agcaggtgct cgtggtcaac caggagaacg tggaaggaca 600

ggagcaccag gacctgctgg tgctagaggt tctgatggat ctacaggtcc aaccggacca 660

gccggaccaa caggttcagc tggaccacca ggttaccctg gagcccctgg tcctaaaggt 720

gaacaaggac ccaccggaaa tccaggacca gctggtccag caggacctag aggagaaacc 780

ggtcaaccag gacaatcagg tcccaccgga cccccaggaa atcctggtgc taatggacaa 840

acaggcgcta aaggagcagc tggacagcct ggtacagctg gtgcccctgg tcaacctgga 900

ccaagaggaa ccccaggacc tgcaggagct gctggtgcta ccggtgcacg tggtcaaaca 960

ggagaacctg gtcctgccgg ttcaaaaggt gaaagtggaa acaagggtga acctggtgct 1020

gcaggacctc agggtccacc aggaccaagt ggtgaggagg gtaaacgtgg tcctaacggt 1080

gaacctggaa gtactggacc agctggacct cccggacaaa gaggttcccc aggttccaga 1140

ggtcaaccag gtgccgatgg tagagctgga acaatgggac ctgccggttc cagaggtgct 1200

actggtccag ctggaaccag aggtcctaac ggagactctg gtaggcctgg tgaacctggt 1260

cagatgggtc cacgtggata tcctggttca cccggaaata cgggtcccgc tggaaaagaa 1320

ggcccaactg gtcaaccagg tacagatggt agaccaggac ccactggacc tgctggagct 1380

agaggtgagc ctggtaatac aggataccca ggtccaaaag gacccacggg agaacctgga 1440

aaaccaggtg ataaaggtca tgctggtcaa gccggagcaa gaggtgctcc aggtccagac 1500

ggaaataacg gagcacaagg accacctggt ccacagggta cccaaggcgg aaaaggtgaa 1560

caaggtccag ctggtccccc cggttaccaa ggtcaacccg gaccagcagg tacggcaggt 1620

gaaacaggta aaccaggtga aagaggacaa ccaggagaat atggccaacc cggtcctgct 1680

ggtgctagag gagagagagg accacctgga gaaagtggag ctgctggacc tgctggtcct 1740

actggaagta gaggaccctc tggaccacca ggaccagacg gtaacaaggg tgagccaggt 1800

acacaaggtg cacctggtac cgctggtcct tccggaccta gtggccaacc aggtgagaga 1860

ggagctgctg gtactcctgg aggtaagggt gagaagggtg aaactggaca aagaggtgag 1920

acaggaaatc ctggcaggga tggagctaga ggagccccag gtgctactgg agcaccaggt 1980

cctgctggag ctaatggcga tcgaggagaa gctggagctg ctggtcctgc aggtccagct 2040

ggacctagag gttctccagg cgaaagagga gaaacaggtc ctgctggccc aaacggatac 2100

gcaggacctg ctggagctgc tggtcagcca ggagctaagg gagaaagagg tactaagggt 2160

ccaaagggcg aaaatggccc tactggtcca actggtccta ccggtgctgc aggtccttca 2220

ggtcctaacg gaccacctgg cccagcaggt tctcgtggag atggtggacc acctggcaca 2280

accggatatc ccggtgctgc tggtcgtact ggtccaccag gacccagtgg tacttcaggt 2340

ccaccaggac caccaggtgc tgctggtaag gaaggccaac gtggtccaag aggcgatcag 2400

ggacctacag gacgagcagg agaaactggt gcaagtggcc caccaggata cgcaggtgaa 2460

aagggaccaa gtggagaacc cggaacagct ggacctccag gtacaccagg accacaaggt 2520

caacagggtg ctccaggtac tcaaggtcag ccaggtagta gaggtgaaag gggtcaacca 2580

ggtaccgcag gttcacaagg agaaccaggt ccacaaggta ctgcaggacc cccaggtgca 2640

agaggtccac caggagctac aggtgcacct ggcaccaacg gagcacctgg tgaagctgga 2700

cgtgacggta atccaggatc cgacggtcca ccaggaaggg acggtcagcc aggacataag 2760

ggtgaacgtg gatatcctgg taatgcagga ccaacaggtg caaccggagc accaggacct 2820

catggaccaa caggcccaac aggaaagcat ggtaacagag gtgagcctgg tccaactgga 2880

tctaccggtc ctaccggagc cactggtcca cgaggaccat caggaccaca aggaactaga 2940

ggtgataaag gagaaccagg tgacaaggga cctagaggac agccaggaac taaaggccac 3000

aatggtcagc aaggacagcc tggccaggca ggtcagcatg gagatcaagg tgccccagga 3060

agtacaggtc ctgccggacc aagaggtcct gctggtccta ctggtccaac tggaaaggac 3120

ggacgtagtg gacaacctgg aacaacaggt ccagctggta ccagaggctc tcaaggttct 3180

caaggtcctg caggaccacc tggtcctcct ggtccaccag gaccaccagg tccttctggt 3240

ggaggctatg actatggtta cgatggtgac tattacaggg ccgaccaacc acgatcaccc 3300

ccatcacaaa gaccaaagga ttacgaaact gatgctacac aaaaatctca aaataaccaa 3360

actgagacac agcagactcc agagggatct cgtaaaaacc cagctagaac atgtagagac 3420

caaagacagt ctcatcctga atggtcatcc ggttattact ggactgatcc taaccaagga 3480

tgtactatgg atgctactaa aacttactgc gactattcta cgggtgagac atgtactaga 3540

gcacaaccag agaacacacc tgccaagaat tggtatagat cttctaaagc aaaaaaacat 3600

acctggcagg gtgaaactac caacggtggt actcagtacg aatataacac agaaggtacc 3660

actactaagg aaatggctac acagcaagct tacatgaggc agcaagccaa tcatgcatct 3720

caaaacacca cctatcactg caagaactct actgcctatc aagatgaaga gactggcaat 3780

caaaagaagg ccacaactca acaaggttca aatgacaccg aacagactgc agagggaaac 3840

tctagataca cgtatactac ccaaaccgat ggatgctctc gaaagactaa cgagtgggga 3900

aagacgacta ccgaatataa aacaaacaaa ccatcccgtc agccaacaca ggataccgct 3960

cagcaagata caggaggtgc cgaccaagag tatggacaag acacaggtcc aacatgttat 4020

aag 4023

Claims

1. A water-soluble collagen comprising an α 1 chain and an α 2 chain; the amino acid sequence of the alpha 1 chain is shown as SEQ ID NO. 1; the amino acid sequence of the alpha 2 chain is shown as SEQ ID NO. 2.

2. The water-soluble collagen according to claim 1, wherein: the molar ratio of the alpha 1 chain to the alpha 2 chain is 2: 1.

3. A gene encoding the water-soluble collagen according to claim 1.

4. The coding gene according to claim 3, characterized in that: the coding gene sequence of the alpha 1 chain is shown as SEQ ID NO. 3; the coding gene sequence of the alpha 2 chain is shown as SEQ ID NO. 4.

5. An expression cassette, vector or host bacterium comprising the coding gene of claim 3 or 4.

6. A method for preparing the water-soluble collagen of claim 1, comprising the steps of: introducing the coding gene of claim 3 or 4 into an expression host bacterium to obtain a recombinant bacterium, culturing the recombinant bacterium and inducing protein expression, and extracting and purifying the protein.

7. An anti-fatigue product comprising the water-soluble collagen of claim 1.

8. The product of claim 7, wherein: the dosage form of the product is capsule, powder, tablet, granule, pill, emulsion, paste or solution.

9. The product of claim 7, wherein: the administration mode of the product is oral administration, injection, instillation or external application.

10. Use of the water-soluble collagen of claim 1 for the preparation of an anti-fatigue product.