CN113880941B

CN113880941B - Recombinant humanized IxIII collagen, expression strain and application thereof

Info

Publication number: CN113880941B
Application number: CN202010631961.8A
Authority: CN
Inventors: 赵健烽; 徐鹏程; 余继刚; 高力虎; 冯丽萍; 黄建民
Original assignee: Jiangsu Jland Biotech Co ltd
Current assignee: Jiangsu Jland Biotech Co ltd
Priority date: 2020-07-03
Filing date: 2020-07-03
Publication date: 2023-11-28
Anticipated expiration: 2040-07-03
Also published as: CN113880941A

Abstract

The application discloses recombinant humanized IxIII collagen, an expression strain and application thereof. The recombinant human source IxIII collagen is formed by splicing hydrophilic fragment recombinant human source type I collagen I69 aa and/or recombinant human source type III collagen III 96 aa. The recombinant humanized IxIII collagen expression strain constructed by the application can effectively and stably express the recombinant humanized IxIII collagen in a large quantity. The recombinant humanized IxIII collagen has good hydrophilicity and stability, and the short peptide structure consisting of 165 amino acids is 100% identical to the corresponding protein sequences in the human collagen I and the human collagen III, so that the recombinant humanized IxIII collagen can not cause immune rejection when applied to a human body. The recombinant human-derived IxIII collagen is mild and does not stimulate, can effectively relieve the problem of facial red blood streaks, plays a role in relieving and repairing, and can be widely applied to the fields of biomedical materials, cosmetics and the like.

Description

Recombinant humanized IxIII collagen, expression strain and application thereof

Technical Field

The application belongs to the technical field of bioengineering bacteria, and relates to recombinant human IxIII collagen, pichia pastoris engineering bacteria for expressing the recombinant human IxIII collagen and application thereof.

Background

Red blood filaments are mainly due to facial phenomena caused by telangiectasia, which are caused by the fact that the capillary vessel position is more easily contacted and perceived by external environment change due to the fact that the facial stratum corneum is weak. The face of the patient looks redder than the skin color of the common normal population, namely the plateau face which is commonly called by people, and only the cheeks on two sides are reddened, and the boundaries are circular and are generally arranged in a silk-like manner. The skin is thin and sensitive, and the color of the skin is redder when supercooling or overheating or emotional agitation. Severe individuals also develop deposited stains that are difficult to cure.

Collagen is a protein widely distributed in connective tissue of human body, and is the protein with the highest content in human body, accounting for 25 to 35 percent of the total protein. Its main functions are maintaining extracellular environment, maintaining normal physiological functions of tissue and organ and repairing injury of body. Collagen is a natural biological resource, has excellent biocompatibility which is incomparable with other high molecular materials, and has supporting elasticity and degradability for cells. Therefore, the collagen can be widely applied to industries such as medicines, cosmetics and the like.

However, natural collagen is insoluble in water, has inhomogeneous properties, is difficult to be used by human body, and often needs to be treated by chemical means before it can be used. In addition, collagen products currently marketed are obtained from animal tissues such as pigs, cows, fish, etc., which are difficult to avoid viral infections and are not compatible with humans, resulting in immune rejection and allergic symptoms. Therefore, the collagen can only be used in cosmetics and health care products, and the original biological functions of the collagen can not be exerted at all.

To solve the problems of conventional animal collagen, many scholars began to apply biotechnology to produce recombinant collagen. The production of collagen by recombinant microorganisms is becoming a mainstream for various convenience in microorganism culture. Fan Dai et al use recombinant human-like collagen produced by high-density fermentation culture of Escherichia coli, and have been successfully applied to the cosmetic field, realizing industrial production (Chinese patent 201310157411.7, 201510883010.9, etc.). However, the bacterial expression system has biological safety problems such as endotoxin, pyrogen and the like, so that the production and detection costs of the product are high, and potential safety hazards exist; the expressed protein exists in bacterial cells in the form of inclusion bodies, so that the purification of the product is difficult and the recovery rate is limited; in addition, the prokaryotic expression system is lower, and post-translational modification of the expression product can not be completed, so that the product has no biological activity.

Thus, more and more research is beginning to utilize eukaryotic microorganisms to produce recombinant collagen. Yeast has natural advantages. Firstly, the yeast has a long history of use in industries such as food, pharmaceutical chemical industry and the like, can be used for secretion expression of exogenous proteins, is beneficial to downstream separation and purification processes, and can not have biological safety problems such as endotoxin, heat sources and the like bacteria.

Disclosure of Invention

The application combines the human type I collagen and the short peptide in the human type III collagen, and the recombinant human collagen with high activity is obtained by repeating the short peptide, so as to provide the recombinant human type IxIII collagen with excellent hydrophilicity, the Pichia pastoris engineering bacteria for secretory expression of the protein and the application thereof, and the extracellular secretory expression of the recombinant human type IxIII collagen can be efficiently and safely carried out.

The technical scheme for realizing the purpose of the application is as follows:

the amino acid sequence of the recombinant human source type I collagen I69 aa is shown as SEQ No. 3.

The amino acid sequence of the recombinant human-derived type III collagen III 96aa is shown as SEQ No. 4.

The recombinant human source IxIII collagen is formed by splicing recombinant human source I type collagen I69 aa and/or recombinant human source III type collagen III 96 aa. Specifically, the recombinant human source type I collagen I69 aa is formed by connecting and splicing a plurality of recombinant human source type I collagen I69 aa end to end; or a plurality of recombinant human source III type collagen III 96aa are connected and spliced end to end; or the recombinant human source type I collagen I69 aa and the recombinant human source type III collagen III 96aa are connected and spliced end to end; or the short peptide formed by splicing the recombinant human I-type collagen I69 aa and the recombinant human III-type collagen III 96aa in an end-to-end connection way is used as a repeating unit, and the repeated short peptide is obtained by repeating for N times, wherein N is more than or equal to 2; or a plurality of recombinant human source type I collagen I69 aa and a plurality of recombinant human source type III collagen III 96aa are connected and spliced end to end; or the short peptide formed by connecting and splicing a plurality of recombinant human source type I collagen I69 aa and a plurality of recombinant human source type III collagen III 96aa end to end is used as a repeating unit, and the repeated short peptide is obtained by repeating N times, wherein N is more than or equal to 2. The number of the components is more than or equal to 2.

In a specific embodiment, the application provides a recombinant human IxIII collagen IxIII 165aa, which is a 165-amino acid short peptide formed by connecting a short peptide sequence of human type I collagen shown in SEQ NO.3 and a short peptide sequence of human type III collagen shown in SEQ NO.4 end to end, wherein the amino acid sequence is shown in SEQ NO. 5.

In a specific embodiment, the application provides a recombinant human IxIII collagen IxIII 495aa, which is a repeated short peptide obtained by repeating 165 amino acids formed by connecting a short peptide sequence of human type I collagen shown in SEQ NO.3 and a short peptide sequence of human type III collagen shown in SEQ NO.4 end to end for 3 times, wherein the amino acid sequence is shown in SEQ NO. 6. Its theoretical molecular weight is about 45.049kd.

The nucleotide sequence of the coding gene of the recombinant human IxIII collagen IxIII 495aa is shown as SEQ No. 7.

The nucleotide sequence of the recombinant human IxIII collagen plasmid ppic9K-IxIII constructed by the application is shown as SEQ No.7, and is constructed by connecting the coding gene of the recombinant human IxIII collagen IxIII 495aa to Xho I and Not I of a ppic9K vector through double enzyme digestion.

The strain for secretory expression of recombinant human IxIII type collagen IxIII 495aa constructed by the application is Pichia pastoris with the preservation number of CGMCC No.16464, which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms on 9 months 12 days of 2018, and the preservation address is North Chen Silu No.1, 3 of the Korean region of Beijing, and the institute of microbiology of China academy of sciences. The Pichia pastoris Pichia pastoris JY0501 is constructed by inducing a linearized recombinant human IxIII type collagen plasmid ppic9K-IxIII obtained by enzyme digestion of SacI into the Pichia pastoris.

The application also provides application of the recombinant humanized IxIII collagen IxIII 495aa in preparing a red blood wire repairing skin care product.

Preferably, in the above-mentioned red blood wire repairing skin care product, the concentration of the recombinant human IxIII collagen IxIII 495aa is 0.2% -0.4% (w/v, g/mL), preferably 0.3% (w/v, g/mL).

The application selects nucleotide sequences with strong water solubility and stability from the spiral regions of human collagen genes I and III with known sequences, inserts the optimized gene fragment into pichia pastoris expression plasmid, converts pichia pastoris to screen to obtain high-expression pichia pastoris genetic engineering bacteria, and obtains high-purity recombinant human collagen through preliminary fermentation and purification steps. The recombinant human ppic9K type collagen has good hydrophilicity and stability, has the structure 100% identical to that of the corresponding part of the natural collagen gene sequence, can not cause immune rejection when applied to human bodies, and has potential application prospects in the fields of biomedical materials, cosmetics and the like.

Drawings

FIG. 1 is a diagram showing the hydrophobicity analysis of human type I alpha 1 chain collagen amino acids.

FIG. 2 is a graph of hydrophobicity analysis of human type III alpha 1 chain collagen amino acids.

FIG. 3 is a diagram showing the hydrophobicity analysis of amino acid IxIII 495aa of recombinant human IxIII collagen.

FIG. 4 is a schematic diagram of the construction of recombinant human IxIII collagen plasmid ppic 9K-IxIII.

FIG. 5 is a Sac I cut agarose gel electrophoresis of recombinant human IxIII collagen plasmid ppic 9K-IxIII.

FIG. 6 is a plate diagram of the electric transformed Pichia pastoris GS 115.

FIG. 7 is a gel electrophoresis pattern of positive clone strains.

FIG. 8 is a PCR gel electrophoresis of positive clone strains.

FIG. 9 is a graph showing protein expression analysis of samples from #5 positive clone strains cultured for 0, 24, 48, 72, and 96 hours.

FIG. 10 is a graph showing protein expression analysis of samples from #6 positive clone strains cultured for 0, 24, 48, 72 and 96 hours.

FIG. 11 is a graph showing protein expression analysis of samples from #7 positive clone strains cultured for 0, 24, 48, 72 and 96 hours.

FIG. 12 is a graph showing protein expression analysis of samples from #8 positive clone strains cultured for 0, 24, 48, 72 and 96 hours.

Fig. 13 is a graph showing the result of moisture improvement rate.

Fig. 14 is an effect result graph.

FIG. 15 is a graph showing the improvement rate of the surface area of red blood filaments.

Fig. 16 is a texture improvement ratio chart.

Fig. 17 is a diagram of the front and rear face situation of a A, B, C group of testers.

Detailed Description

The application is further described in detail below with reference to examples and figures. The procedures, conditions, reagents, experimental methods, etc. for carrying out the present application are common knowledge and common knowledge in the art, except for those specifically mentioned below, and the present application is not particularly limited.

Example 1

1. Protein sequence selection

The amino acid sequence (SEQ No. 1) of the human type I alpha 1 chain collagen and the amino acid sequence (SEQ No. 2) of the human type III alpha 1 chain collagen are subjected to hydrophobicity analysis, and the evaluation results are shown in figures 1 and 2. The lower the hydrophobicity score, the better its hydrophilicity. According to the hydrophobicity analysis result, selecting the amino acid fragments with low scores, taking the 69 amino acid short peptide fragments of the type I collagen (i.e. recombinant human type I collagen I69 aa and SEQ No. 3) and the 96 amino acid short peptide fragments of the type III collagen (recombinant human type III collagen III 96aa and SEQ No. 4), connecting the two short peptide fragments into 165 short peptide fragment units (i.e. recombinant human type IxIII collagen IxIII 165aa and SEQ No. 5), and repeating the fragment units end to end for three times to integrate into a novel protein, namely the recombinant human type IxIII collagen (i.e. recombinant human type IxIII collagen IxIII 495aa and SEQ No. 6). The amino acid hydrophobicity analysis of the recombinant humanized IxIII type collagen shows that the hydrophobicity evaluation of all amino acids in the protein is lower than zero as shown in the figure 3, which shows that the protein has good hydrophilicity.

2. Plasmid construction and linearization

Translation of recombinant human type IxIII 495aa into a base sequence (SEQ No. 7), synthesis of gene by PAS (PCR-based Accurate Synthesis) method, double cleavage between XhoI and Not I of IxIII 495aa to ppic9K vector, and FIG. 4 is a schematic diagram of construction of recombinant human type IxIII collagen plasmid ppic 9K-IxIII. Transferring the obtained recombinant plasmid ppic9K-IxIII into TOP10 clone strain, picking positive clone for sequencing, and splicing the sequencing result as shown in SEQ No. 8. The region between 244 th base and 249 th base and between 1756 th base and 1763 th base of SEQ No.8 is enzyme cutting site.

20 mug of plasmid was extracted, and the plasmid was subjected to tangential digestion with SacI enzyme, and lyophilized and concentrated for use. Digested for 3h at 37 ℃. mu.L was subjected to 1% agarose gel electrophoresis, and the result of the electrophoresis is shown in FIG. 5. Wherein M is a DNA standard substance, 1000, 2000, 3000, 4000, 5000, 6000, 8000, 10000bp from bottom to top; 1 is Sac I enzyme cutting; 2 is the recovered target fragment.

TABLE 1 Table for preparing enzyme tangential system

3. Electric transfer Pichia pastoris cell GS115

Ice bath electric cup, 10. Mu.L of linearized plasmid was added to a 1.5mL EP tube containing 80. Mu.L of Pichia pastoris GS115 competent cells, mixed well and transferred to an electric cup with a diameter of 0.2cm, and then the electric cup was ice-bathed for 5min. The electric shock conditions are as follows: the voltage is 1.5kV; capacitance 25 μF; the resistance is 200 omega, and the electric shock time is 4-10 msec. After the electric shock is completed, 650uL of sorbitol solution precooled on ice and with the concentration of 1M is added into an electric shock conversion cup, and the solution is gently blown by a gun head to be uniform. All the liquid in the electrorotor was transferred to a new 2ml EP tube and incubated at 30℃for 2h. The bacterial cells are collected by low-speed centrifugation and are all coated on an MD plate, and are cultivated for 3-4d at the constant temperature of 30 ℃. FIG. 5 is a plate diagram of the electric transformed Pichia pastoris GS 115.

Identification of Positive clone Strain by PCR

After the colony grows out of the plate, picking up the single bacteria growing on the plate by an inoculating loop, and inoculating the single bacteria into a centrifuge tube filled with 500 mu L of YPD liquid culture medium, and culturing at 30 ℃ for 180r/min overnight. 10 clones were selected and genomic DNA was extracted, respectively, as shown in FIG. 6. In the figure, M is a DNA standard substance, 1000, 2000, 3000, 4000, 5000, 6000, 8000 and 10000bp from bottom to top; 1-10: genome extracted from each clone strain.

PCR using primers on the vector identified that the expected band size was approximately 2kb,2kb being the amplified sequence size, the results are shown in FIG. 7, where M: DNA standard, from bottom to top, 100, 200, 500, 750, 1000, 2000bp;1-10: PCR amplified fragments of each clone strain.

5. Small test expression

Induction of expression: taking 50 μl of identified positive strain (5#, 6#,7#, 8#) and inoculating into an conical flask containing 10ml BMGY, culturing at 30deg.C for 220r/min overnight, shaking to OD 600=2-6 (logarithmic growth, about 16-18 h); centrifuging at room temperature for 5min at 5000r/min, collecting cells, removing supernatant, and re-suspending cells with 10ml BMMY for induction expression; 1mL of the culture medium was sampled every 24h, and methanol was added to a final concentration of 0.5% for continued induction; at each of time points 0, 24, 48, 72, 96hr samples were centrifuged at 10000r/min for 2min and supernatants were collected for detection.

Concentrating the expression product by trichloroacetic acid precipitation method:

(1) Adding 500 mu L of culture solution supernatant and 1/9 volume of 100% TCA into a centrifuge tube, mixing by shaking, and precipitating at 4 ℃ overnight;

(2) Centrifuging at 12000r/min for 10min, precipitating into viscous yellowish brown jelly, discarding supernatant, collecting precipitate, inverting the EP tube on absorbent paper, and standing in an oven at 37deg.C for 10-20 min to make the tube wall have no obvious liquid residue;

(3) Adding 200 mu L of cold acetone, shaking and mixing uniformly, standing a sample at room temperature for 10min, and washing away TCA remained on the tube wall and the tube bottom;

(4) Centrifuging for 10min at 12000r/min, discarding supernatant, repeating the steps (2) and (3) for 2-3 times;

(5) Adding 30 mu L of loading buffer solution, incubating for 1h at 37 ℃, and dissolving the precipitate; if the precipitate is not dissolved, it can be blown with a 100. Mu.L gun head until the precipitate is dissolved.

SDS-PAGE electrophoresis detection: the analysis results of the expression conditions of the 5# positive bacteria, the 6# positive bacteria and the 7# positive bacteria and the 8# positive bacteria are shown in figures 8, 9, 10 and 11. Wherein M: a protein standard; 1: the GS115 strain was cultured for 72 hours to obtain a supernatant; 2: positive strain was cultured for 0 hours supernatant; 3: positive strain was cultured for 24 hours supernatant; 4: positive strain culture for 48 hours supernatant; 5: positive strain was cultured for 72 hours supernatant; 6: positive strains were cultured for 96 hours supernatant.

Taking 8# positive bacteria for strain preservation, namely Pichia pastoris Pichia pastoris JY0501, wherein the strain is preserved in China general microbiological culture Collection center (preservation center address: north Xiyu No.1, 3 of the North Chen West Lu No.1 of the Chaiyang area of Beijing city, and postal code: 100101) in 9 months 12 days of 2018, and the preservation number is CGMCC No.16464.

The application selects nucleotide sequences with good water solubility and strong stability from the spiral regions of human collagen genes I and III with known sequences, inserts the optimized gene fragment into pichia pastoris expression plasmid, converts pichia pastoris to screen and obtain high-expression pichia pastoris genetic engineering bacteria, and obtains high-purity recombinant human collagen through preliminary fermentation and purification steps. Experiments prove that the recombinant human-like collagen produced by the method has good hydrophilicity and stability, and the structure of the recombinant human-like collagen is 100 percent identical to that of the corresponding part of the natural collagen gene sequence, so that the recombinant human-like collagen can not cause immune rejection when applied to human bodies, and can be widely applied to the fields of biomedical materials, cosmetics and the like. The application adopts the secretion type expression vector, successfully realizes the secretion type expression of the recombinant human-like collagen, and the expression product is secreted in the supernatant, thereby being convenient for purification, having the advantages which are not possessed by other recombinant human-like collagen production, and being convenient for large-scale production operation. After the vector is electrically transferred into pichia pastoris, the gene is integrated on the genome of the pichia pastoris, so that the stability of the recombinant bacterium is good, the gene is not easy to run off after multiple passages, the character of high-efficiency expression can be kept, stable production can be well realized, the production method of the pichia pastoris is aerobic fermentation, the thallus density is high, and the expression quantity is also greatly improved.

Example 2

1. Selecting 150 persons, randomly dividing the persons into 3 groups, 50 persons in each group, finally determining that the effective number is 40 persons, cleaning the face every night, taking recombinant humanized IxIII collagen IxIII 495aa skin care liquid for use, using an instrument 4 times before, 2 days after use, 11 days and 15 days, photographing a subject each time, analyzing the effect, and analyzing the surface area of red blood wires and the texture of the skin by using an Antera3D skin tester (Ireland).

2. Effect determination

Effect= (pre-treatment skin damage count-post-treatment skin damage count)/pre-treatment skin damage count 100%.

Effect level:

the effect is shown: the redness and swelling or stinging subsides by 60 to 90 percent, and the symptoms are relieved;

the method is effective: the redness and swelling or stinging subsides by 20 to 60 percent, and the symptoms are improved;

invalidation: the redness and swelling or stinging subsided < 20%, and the clinical symptoms were not improved.

The experiment is divided into a group A, a group B and a group C, and the recombinant human source IxIII collagen IxIII 495aa freeze-dried cake with the same formula and the same batch is used as a matrix, and each bottle is 5mg. In order to ensure the authenticity of the experiment, the group A and the group C are control groups, and the concentration of the recombinant human IxIII collagen IxIII 495aa is 0.1% (w/v, g/mL, namely 1 g/L); group C, recombinant human IxIII collagen IxIII 495aa concentration of 0.5% (w/v, g/mL, i.e., 5 g/L), group B, experimental group, recombinant human IxIII collagen IxIII 495aa concentration of 0.3% (w/v, g/mL, i.e., 3 g/L).

The 4 skin index test data for groups a, B and C are shown in the table below.

TABLE 2 detection results of 0.1% recombinant humanized IxIII collagen IxIII 495aa from group A

After 15 days of use of 0.1% recombinant human IxIII collagen IxIII 495aa, the test results showed that:

-moisture: the percentage moisture improvement was 3.49% compared to before use.

-effect: compared with the prior use, the repair effect of the recombinant human IxIII collagen IxIII 495aa with low concentration on red blood filaments is only improved by 54.55 percent.

Texture: each time point is statistically significant.

TABLE 3 detection results of 0.3% recombinant humanized IxIII collagen IxIII 495aa from group B

After 15 days of use of 0.3% recombinant human IxIII collagen IxIII 495aa, the test results showed that:

-moisture: the product has a moisturizing effect for 15 days, and the moisture improvement percentage is 5.44% compared with the product before use.

-effect: compared with the prior use, the effect is greatly improved in 15 days, the improvement percentage reaches 79.80 percent, the surface area of the red blood wires is reduced, and the same ratio is reduced by 53.13 percent.

Texture: has statistical significance.

When the concentration of the recombinant human IxIII collagen IxIII 495aa is 0.2% -0.4%, the repair effect on red blood filaments is similar, and the application takes 0.3% of recombinant human IxIII collagen IxIII 495aa as a representative example.

TABLE 4 detection results of 0.5% recombinant humanized IxIII collagen IxIII 495aa from group C

After 15 days of use of 0.5% recombinant human IxIII collagen IxIII 495aa, the test results showed that:

moisture content: the percentage moisture improvement was 4.90% compared to before use.

The effect is as follows: the high-concentration recombinant human-source IxIII collagen IxIII 495aa plays a certain role in repairing the red blood wires, but the surface area of the red blood wires is reduced by 40.40 percent.

Texture: the P value is less than 0.01, and has statistical significance.

In summary, according to the three A, B, C groups, through continuous use of 15 days of testers, four aspects of moisture, effect, red blood streak surface area and texture are analyzed for 4 times, and the detection result shows that the recombinant humanized IxIII collagen IxIII 495aa with the concentration of 0.2% -0.4% is mild and not stimulated, so that the facial red blood streak problem can be effectively relieved, and the effect of relieving and repairing is achieved.

Sequence listing

<110> Jiangsu Jiangshan Zhiyuan biotechnology Co., ltd

<120> recombinant humanized IxIII collagen, expression strain and use thereof

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Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp

1300 1305 1310

Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu

1315 1320 1325

Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp

1330 1335 1340

Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr

1345 1350 1355 1360

Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr

1365 1370 1375

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

1380 1385 1390

Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr

1395 1400 1405

Ser Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys

1410 1415 1420

Thr Val Ile Glu Tyr Lys Thr Thr Lys Thr Ser Arg Leu Arg Ile Ile

1425 1430 1435 1440

<210> 2

<211> 1466

<212> PRT

<213> Homo sapiens

<400> 2

Met Met Ser Phe Val Gln Lys Gly Ser Trp Leu Leu Leu Ala Leu Leu

1 5 10 15

His Pro Thr Ile Ile Leu Ala Gln Gln Glu Ala Val Glu Gly Gly Cys

20 25 30

Ser His Leu Gly Gln Ser Tyr Ala Asp Arg Asp Val Trp Lys Pro Glu

35 40 45

Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Ser Val Leu Cys Asp Asp

50 55 60

Ile Ile Cys Asp Asp Gln Glu Leu Asp Cys Pro Asn Pro Glu Ile Pro

65 70 75 80

Phe Gly Glu Cys Cys Ala Val Cys Pro Gln Pro Pro Thr Ala Pro Thr

85 90 95

Arg Pro Pro Asn Gly Gln Gly Pro Gln Gly Pro Lys Gly Asp Pro Gly

100 105 110

Pro Pro Gly Ile Pro Gly Arg Asn Gly Asp Pro Gly Ile Pro Gly Gln

115 120 125

Pro Gly Ser Pro Gly Ser Pro Gly Pro Pro Gly Ile Cys Glu Ser Cys

130 135 140

Pro Thr Gly Pro Gln Asn Tyr Ser Pro Gln Tyr Asp Ser Tyr Asp Val

145 150 155 160

Lys Ser Gly Val Ala Val Gly Gly Leu Ala Gly Tyr Pro Gly Pro Ala

165 170 175

Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Thr Ser Gly His Pro Gly

180 185 190

Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln

195 200 205

Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser

210 215 220

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

225 230 235 240

Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile

245 250 255

Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn

260 265 270

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

275 280 285

Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala

290 295 300

Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg

305 310 315 320

Gly Asn Asp Gly Ala Arg Gly Ser Asp Gly Gln Pro Gly Pro Pro Gly

325 330 335

Pro Pro Gly Thr Ala Gly Phe Pro Gly Ser Pro Gly Ala Lys Gly Glu

340 345 350

Val Gly Pro Ala Gly Ser Pro Gly Ser Asn Gly Ala Pro Gly Gln Arg

355 360 365

Gly Glu Pro Gly Pro Gln Gly His Ala Gly Ala Gln Gly Pro Pro Gly

370 375 380

Pro Pro Gly Ile Asn Gly Ser Pro Gly Gly Lys Gly Glu Met Gly Pro

385 390 395 400

Ala Gly Ile Pro Gly Ala Pro Gly Leu Met Gly Ala Arg Gly Pro Pro

405 410 415

Gly Pro Ala Gly Ala Asn Gly Ala Pro Gly Leu Arg Gly Gly Ala Gly

420 425 430

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

435 440 445

Arg Gly Glu Ala Gly Ile Pro Gly Val Pro Gly Ala Lys Gly Glu Asp

450 455 460

Gly Lys Asp Gly Ser Pro Gly Glu Pro Gly Ala Asn Gly Leu Pro Gly

465 470 475 480

Ala Ala Gly Glu Arg Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly Pro

485 490 495

Asn Gly Ile Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro

500 505 510

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

515 520 525

Val Pro Gly Gly Pro Gly Met Arg Gly Met Pro Gly Ser Pro Gly Gly

530 535 540

Pro Gly Ser Asp Gly Lys Pro Gly Pro Pro Gly Ser Gln Gly Glu Ser

545 550 555 560

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

565 570 575

Val Met Gly Phe Pro Gly Pro Lys Gly Asn Asp Gly Ala Pro Gly Lys

580 585 590

Asn Gly Glu Arg Gly Gly Pro Gly Gly Pro Gly Pro Gln Gly Pro Pro

595 600 605

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

610 615 620

Pro Gly Gly Asp Lys Gly Asp Thr Gly Pro Pro Gly Pro Gln Gly Leu

625 630 635 640

Gln Gly Leu Pro Gly Thr Gly Gly Pro Pro Gly Glu Asn Gly Lys Pro

645 650 655

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

660 665 670

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

675 680 685

Ala Gly Ala Pro Gly Leu Arg Gly Gly Ala Gly Pro Pro Gly Pro Glu

690 695 700

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

705 710 715 720

Thr Pro Gly Leu Gln Arg Met Pro Gly Glu Arg Gly Gly Leu Gly Ser

725 730 735

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

740 745 750

Gly Val Pro Gly Lys Asp Gly Pro Arg Gly Pro Thr Gly Pro Ile Gly

755 760 765

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

770 775 780

Pro Gly Leu Pro Gly Ile Ala Gly Pro Arg Gly Ser Pro Gly Glu Arg

785 790 795 800

Gly Glu Thr Gly Pro Pro Gly Pro Ala Gly Phe Pro Gly Ala Pro Gly

805 810 815

Gln Asn Gly Glu Pro Gly Gly Lys Gly Glu Arg Gly Ala Pro Gly Glu

820 825 830

Lys Gly Glu Gly Gly Pro Pro Gly Val Ala Gly Pro Pro Gly Gly Ser

835 840 845

Gly Pro Ala Gly Pro Pro Gly Pro Gln Gly Val Lys Gly Glu Arg Gly

850 855 860

Ser Pro Gly Gly Pro Gly Ala Ala Gly Phe Pro Gly Ala Arg Gly Leu

865 870 875 880

Pro Gly Pro Pro Gly Ser Asn Gly Asn Pro Gly Pro Pro Gly Pro Ser

885 890 895

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

900 905 910

Ala Pro Gly Ser Pro Gly Val Ser Gly Pro Lys Gly Asp Ala Gly Gln

915 920 925

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

930 935 940

Gly Pro Leu Gly Ile Ala Gly Ile Thr Gly Ala Arg Gly Leu Ala Gly

945 950 955 960

Pro Pro Gly Met Pro Gly Pro Arg Gly Ser Pro Gly Pro Gln Gly Val

965 970 975

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

980 985 990

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

995 1000 1005

Glu Pro Gly Arg Asp Gly Asn Pro Gly Ser Asp Gly Leu Pro Gly Arg

1010 1015 1020

Asp Gly Ser Pro Gly Gly Lys Gly Asp Arg Gly Glu Asn Gly Ser Pro

1025 1030 1035 1040

Gly Ala Pro Gly Ala Pro Gly His Pro Gly Pro Pro Gly Pro Val Gly

1045 1050 1055

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

1060 1065 1070

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

1075 1080 1085

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

1090 1095 1100

Ile Lys Gly His Arg Gly Phe Pro Gly Asn Pro Gly Ala Pro Gly Ser

1105 1110 1115 1120

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

1125 1130 1135

Gly Pro Arg Gly Pro Val Gly Pro Ser Gly Pro Pro Gly Lys Asp Gly

1140 1145 1150

Thr Ser Gly His Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Asn

1155 1160 1165

Arg Gly Glu Arg Gly Ser Glu Gly Ser Pro Gly His Pro Gly Gln Pro

1170 1175 1180

Gly Pro Pro Gly Pro Pro Gly Ala Pro Gly Pro Cys Cys Gly Gly Val

1185 1190 1195 1200

Gly Ala Ala Ala Ile Ala Gly Ile Gly Gly Glu Lys Ala Gly Gly Phe

1205 1210 1215

Ala Pro Tyr Tyr Gly Asp Glu Pro Met Asp Phe Lys Ile Asn Thr Asp

1220 1225 1230

Glu Ile Met Thr Ser Leu Lys Ser Val Asn Gly Gln Ile Glu Ser Leu

1235 1240 1245

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

1250 1255 1260

Leu Lys Phe Cys His Pro Glu Leu Lys Ser Gly Glu Tyr Trp Val Asp

1265 1270 1275 1280

Pro Asn Gln Gly Cys Lys Leu Asp Ala Ile Lys Val Phe Cys Asn Met

1285 1290 1295

Glu Thr Gly Glu Thr Cys Ile Ser Ala Asn Pro Leu Asn Val Pro Arg

1300 1305 1310

Lys His Trp Trp Thr Asp Ser Ser Ala Glu Lys Lys His Val Trp Phe

1315 1320 1325

Gly Glu Ser Met Asp Gly Gly Phe Gln Phe Ser Tyr Gly Asn Pro Glu

1330 1335 1340

Leu Pro Glu Asp Val Leu Asp Val His Leu Ala Phe Leu Arg Leu Leu

1345 1350 1355 1360

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

1365 1370 1375

Ala Tyr Met Asp Gln Ala Ser Gly Asn Val Lys Lys Ala Leu Lys Leu

1380 1385 1390

Met Gly Ser Asn Glu Gly Glu Phe Lys Ala Glu Gly Asn Ser Lys Phe

1395 1400 1405

Thr Tyr Thr Val Leu Glu Asp Gly Cys Thr Lys His Thr Gly Glu Trp

1410 1415 1420

Ser Lys Thr Val Phe Glu Tyr Arg Thr Arg Lys Ala Val Arg Leu Pro

1425 1430 1435 1440

Ile Val Asp Ile Ala Pro Tyr Asp Ile Gly Gly Pro Asp Gln Glu Phe

1445 1450 1455

Gly Val Asp Val Gly Pro Val Cys Phe Leu

1460 1465

<210> 3

<211> 69

<212> PRT

<213> Homo sapiens

<400> 3

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

1 5 10 15

Asp Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Gly Leu Asn

65

<210> 4

<211> 96

<212> PRT

<213> Homo sapiens

<400> 4

Gly Ala Ala Gly Glu Arg Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly

1 5 10 15

Pro Asn Gly Ile Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala

20 25 30

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

35 40 45

Gly Val Pro Gly Gly Pro Gly Met Arg Gly Met Pro Gly Ser Pro Gly

50 55 60

Gly Pro Gly Ser Asp Gly Lys Pro Gly Pro Pro Gly Ser Gln Gly Glu

65 70 75 80

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

85 90 95

<210> 5

<211> 165

<212> PRT

<213> Homo sapiens

<400> 5

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

1 5 10 15

Asp Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Gly Leu Asn Gly Ala Ala Gly Glu Arg Gly Ala Pro Gly Phe

65 70 75 80

Arg Gly Pro Ala Gly Pro Asn Gly Ile Pro Gly Glu Lys Gly Pro Ala

85 90 95

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

100 105 110

Glu Pro Gly Arg Asp Gly Val Pro Gly Gly Pro Gly Met Arg Gly Met

115 120 125

Pro Gly Ser Pro Gly Gly Pro Gly Ser Asp Gly Lys Pro Gly Pro Pro

130 135 140

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

145 150 155 160

Pro Arg Gly Gln Pro

165

<210> 6

<211> 495

<212> PRT

<213> Homo sapiens

<400> 6

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

1 5 10 15

Asp Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Gly Leu Asn Gly Ala Ala Gly Glu Arg Gly Ala Pro Gly Phe

65 70 75 80

Arg Gly Pro Ala Gly Pro Asn Gly Ile Pro Gly Glu Lys Gly Pro Ala

85 90 95

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

100 105 110

Glu Pro Gly Arg Asp Gly Val Pro Gly Gly Pro Gly Met Arg Gly Met

115 120 125

Pro Gly Ser Pro Gly Gly Pro Gly Ser Asp Gly Lys Pro Gly Pro Pro

130 135 140

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

145 150 155 160

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

165 170 175

Thr Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly His Arg Gly Phe Ser

180 185 190

Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly

195 200 205

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

210 215 220

Ala Gly Ala Pro Gly Lys Asp Gly Leu Asn Gly Ala Ala Gly Glu Arg

225 230 235 240

Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly Pro Asn Gly Ile Pro Gly

245 250 255

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

260 265 270

Arg Gly Ala Ala Gly Glu Pro Gly Arg Asp Gly Val Pro Gly Gly Pro

275 280 285

Gly Met Arg Gly Met Pro Gly Ser Pro Gly Gly Pro Gly Ser Asp Gly

290 295 300

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

305 310 315 320

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

325 330 335

Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly

340 345 350

His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser

355 360 365

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

370 375 380

Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Lys Asp Gly Leu Asn Gly

385 390 395 400

Ala Ala Gly Glu Arg Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly Pro

405 410 415

Asn Gly Ile Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro

420 425 430

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

435 440 445

Val Pro Gly Gly Pro Gly Met Arg Gly Met Pro Gly Ser Pro Gly Gly

450 455 460

Pro Gly Ser Asp Gly Lys Pro Gly Pro Pro Gly Ser Gln Gly Glu Ser

465 470 475 480

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

485 490 495

<210> 7

<211> 1485

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ggtccacaag gtccaagagg tgataagggt gaaactggtg aacaaggtga cagaggtatc 60

aagggtcaca gaggtttctc tggattgcaa ggtccacctg gtccaccagg ttctccaggt 120

gagcaaggtc cttctggtgc ttctggtcct gctggaccaa gaggtcctcc aggatctgct 180

ggtgctccag gtaaagatgg tttgaatggt gctgctggtg aaagaggtgc tcctggtttt 240

agaggacctg ctggtcctaa tggtatccca ggtgaaaagg gtccagccgg tgaacgtggt 300

gcacctggac ctgcaggccc tagaggtgct gcaggcgaac ctggtagaga tggtgttcca 360

ggtggtccag gtatgagagg tatgcctggt tctcctggtg gtcctggttc tgatggtaaa 420

ccaggtcctc ctggctctca aggtgaatct ggtagacccg gacctccagg tccaagtggt 480

cctagaggac aacctggtcc tcaaggacct cgtggtgaca aaggtgaaac aggcgagcag 540

ggtgatcgtg gtattaaggg acatagagga ttttccggtc tgcagggacc tccaggacct 600

cctggtagcc ctggtgaaca gggaccatca ggtgctagtg gtcctgccgg tcctcgtggc 660

ccacctggtt cagctggtgc ccctggaaag gatggactta acggtgcagc tggcgaaagg 720

ggagcacccg gattcagagg tccagctggc ccaaacggta ttcctggtga gaaaggacct 780

gccggtgaga ggggtgcacc aggtcctgct ggtcccaggg gtgcagctgg tgaacccggt 840

cgtgatggtg tacctggcgg acctggaatg cgtggtatgc caggcagtcc aggtggccct 900

ggaagtgatg gtaagcccgg tccaccagga tcacagggtg agtcaggtcg tcctggtcct 960

ccaggtcctt ccggtcctag aggtcagcca ggtccacagg gtccccgtgg cgacaaaggc 1020

gagactggcg aacaaggcga taggggaatc aaaggtcata ggggttttag cggacttcaa 1080

ggccctcctg gacctccagg ctcaccaggc gaacaaggtc catccggtgc atcaggccca 1140

gccggtccaa ggggccctcc aggtagtgct ggcgcacctg gtaaagacgg cctaaatggt 1200

gccgcaggcg agagaggcgc tccaggtttc aggggtcccg ccggaccaaa tggaataccc 1260

ggtgaaaaag gccctgctgg cgagcgtggt gccccaggac cagccggacc acgtggtgcc 1320

gccggtgagc caggacgtga cggtgtcccc ggtggtcccg gcatgcgtgg aatgcctgga 1380

tcaccaggcg gtcccggatc agatggaaaa cccggtccac ctggtagtca gggtgaaagt 1440

ggtagacctg gtcctcctgg tccatctgga cctcgtggcc aacca 1485

<210> 8

<211> 1775

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagcttac gtagaattcg gtccacaagg tccaagaggt 300

gataagggtg aaactggtga acaaggtgac agaggtatca agggtcacag aggtttctct 360

ggattgcaag gtccacctgg tccaccaggt tctccaggtg agcaaggtcc ttctggtgct 420

tctggtcctg ctggaccaag aggtcctcca ggatctgctg gtgctccagg taaagatggt 480

ttgaatggtg ctgctggtga aagaggtgct cctggtttta gaggacctgc tggtcctaat 540

ggtatcccag gtgaaaaggg tccagccggt gaacgtggtg cacctggacc tgcaggccct 600

agaggtgctg caggcgaacc tggtagagat ggtgttccag gtggtccagg tatgagaggt 660

atgcctggtt ctcctggtgg tcctggttct gatggtaaac caggtcctcc tggctctcaa 720

ggtgaatctg gtagacccgg acctccaggt ccaagtggtc ctagaggaca acctggtcct 780

caaggacctc gtggtgacaa aggtgaaaca ggcgagcagg gtgatcgtgg tattaaggga 840

catagaggat tttccggtct gcagggacct ccaggacctc ctggtagccc tggtgaacag 900

ggaccatcag gtgctagtgg tcctgccggt cctcgtggcc cacctggttc agctggtgcc 960

cctggaaagg atggacttaa cggtgcagct ggcgaaaggg gagcacccgg attcagaggt 1020

ccagctggcc caaacggtat tcctggtgag aaaggacctg ccggtgagag gggtgcacca 1080

ggtcctgctg gtcccagggg tgcagctggt gaacccggtc gtgatggtgt acctggcgga 1140

cctggaatgc gtggtatgcc aggcagtcca ggtggccctg gaagtgatgg taagcccggt 1200

ccaccaggat cacagggtga gtcaggtcgt cctggtcctc caggtccttc cggtcctaga 1260

ggtcagccag gtccacaggg tccccgtggc gacaaaggcg agactggcga acaaggcgat 1320

aggggaatca aaggtcatag gggttttagc ggacttcaag gccctcctgg acctccaggc 1380

tcaccaggcg aacaaggtcc atccggtgca tcaggcccag ccggtccaag gggccctcca 1440

ggtagtgctg gcgcacctgg taaagacggc ctaaatggtg ccgcaggcga gagaggcgct 1500

ccaggtttca ggggtcccgc cggaccaaat ggaatacccg gtgaaaaagg ccctgctggc 1560

gagcgtggtg ccccaggacc agccggacca cgtggtgccg ccggtgagcc aggacgtgac 1620

ggtgtccccg gtggtcccgg catgcgtgga atgcctggat caccaggcgg tcccggatca 1680

gatggaaaac ccggtccacc tggtagtcag ggtgaaagtg gtagacctgg tcctcctggt 1740

ccatctggac ctcgtggcca accataagcg gccgc 1775

Claims

1. The recombinant human IxIII collagen is characterized in that the amino acid sequence of the recombinant human IxIII collagen IxIII 495aa is shown in SEQ No. 6.

2. The recombinant human-derived IxIII collagen encoding gene according to claim 1, wherein the nucleotide sequence is shown in SEQ No. 7.

3. Recombinant human IxIII collagen plasmid ppic9K-IxIII 495aa, which is characterized in that the nucleotide sequence is shown in SEQ No. 8; the construction method of the recombinant human IxIII collagen plasmid ppic9K-IxIII 495aa is that the coding gene of the recombinant human IxIII collagen IxIII 495aa is constructed between XhoI and Not I which are connected to a ppic9K vector through double enzyme digestion.

4. Recombinant human IxIII collagen expression strain, which is characterized by being Pichia pastorisPichia pastorisJY0501 with preservation number of CGMCC No.16464.

5. Use of recombinant human IxIII collagen IxIII 495aa according to claim 1 in the preparation of a red blood wire skin care product.

6. The use according to claim 5, wherein in the red blood wire repair skin care product, the concentration of recombinant human IxIII collagen IxIII 495aa is 0.2% -0.4% w/v.

7. The use according to claim 5, wherein the concentration of recombinant human IxIII collagen IxIII 495aa in the red blood wire repair skin care product is 0.3% w/v.