阅读说明：本技术 重组人源IxIII胶原蛋白、表达菌株及其应用 (Recombinant human IxIII collagen, expression strain and application thereof ) 是由 赵健烽 徐鹏程 余继刚 高力虎 冯丽萍 黄建民 于 2020-07-03 设计创作，主要内容包括：本发明公开了一种重组人源IxIII胶原蛋白、表达菌株及其应用。所述的重组人源IxIII胶原蛋白由亲水片段重组人源I型胶原蛋白I 69aa和/或重组人源III型胶原蛋白III 96aa拼接而成。本发明构建的重组人源IxIII胶原蛋白表达菌株能够有效稳定和大量地表达重组人源IxIII胶原蛋白。本发明的重组人源IxIII胶原蛋白具有良好的亲水性及稳定性,其165个氨基酸组成的短肽结构与人胶原蛋白I及人胶原蛋白III中对应的蛋白序列100％相同,应用于人体中不会造成免疫排斥。重组人源IxIII胶原蛋白温和不刺激,能够有效缓解面部红血丝问题,起到舒缓修复的作用,可以广泛应用于生物医用材料、化妆品等领域。(The invention discloses a recombinant human IxIII collagen, an expression strain and application thereof. The recombinant human IxIII collagen is formed by splicing a hydrophilic fragment recombinant human I type collagen I69 aa and/or recombinant human III type collagen III 96 aa. The recombinant human IxIII collagen expression strain constructed by the invention can effectively, stably and massively express the recombinant human IxIII collagen. The recombinant human IxIII collagen has good hydrophilicity and stability, the short peptide structure consisting of 165 amino acids is 100% identical to the corresponding protein sequences in human collagen I and human collagen III, and the recombinant human IxIII collagen can not cause immunological rejection when being applied to a human body. The recombinant human IxIII collagen is mild and non-irritating, can effectively relieve the problem of facial red blood streak, plays a role in relieving and repairing, and can be widely applied to the fields of biomedical materials, cosmetics and the like.)

1. The amino acid sequence of the recombinant human III type collagen III 96aa is shown in SEQ No. 4.

2. The recombinant human IxIII collagen is characterized by being formed by splicing a recombinant human I-type collagen I69 aa and/or a recombinant human III-type collagen III 96aa, wherein the amino acid sequence of the recombinant human I-type collagen I69 aa is shown in SEQ No. 3.

3. The recombinant human IxIII collagen according to claim 2, which is formed by splicing a plurality of recombinant human type I collagen I69 aa end to end; or a plurality of recombinant human III-type collagen III 96aa are 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 spliced end to end; or the short peptide formed by splicing the recombinant human source type I collagen I69 aa and the recombinant human source type III collagen III 96aa in an end-to-end connection manner is used as a repeating unit, and the repeating short peptide is obtained by repeating N times, wherein N is more than or equal to 2; or a plurality of recombinant human type I collagen I69 aa and a plurality of recombinant human type III collagen III 96aa are 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 repeating short peptide is obtained by repeating N times, wherein N is more than or equal to 2.

4. The recombinant human IxIII collagen according to claim 2, wherein the amino acid sequence of the recombinant human IxIII collagen IxIII 165aa is shown in SEQ No. 5.

5. The recombinant human IxIII collagen according to claim 2, wherein the amino acid sequence of the recombinant human IxIII collagen IxIII 495aa is shown in SEQ No. 6.

6. The recombinant human IxIII collagen IxIII 495aa encoding gene of claim 5, having a nucleotide sequence shown in SEQ No. 7.

7. The recombinant human IxIII collagen plasmid ppic9K-IxIII 495aa has a nucleotide sequence shown as SEQ No. 8; the construction method of the recombinant human IxIII collagen plasmid ppic9K-IxIII 495aa is constructed by connecting the coding gene of the recombinant human IxIII collagen IxIII 495aa to XhoI and Not I of ppic9K vector through double enzyme digestion.

8. The recombinant human IxIII collagen expression strain is Pichia pastoris JY0501 with the preservation number of CGMCC No. 16464.

9. Application of recombinant human IxIII collagen IxIII 495aa in preparing a red blood cell repairing skin care product.

10. The use of claim 9, wherein the recombinant human IxIII collagen IxIII 495aa is present in the skin care product for repairing red blood filaments at a concentration of 0.2% to 0.4% w/v, preferably 0.3% w/v.

Technical Field

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

Background

The red blood streak is mainly caused by facial phenomenon caused by the fact that the position of capillary vessels is easy to contact and sense the change of external environment due to the weakness of the stratum corneum of the face, and therefore the capillary vessels are expanded. The face of the patient looks redder than the skin color of the general normal population, namely the common plateau face of people, some of the face looks reddish only at cheeks at two sides, and the boundary is circular and generally arranged in a thread shape. The skin is thin and sensitive, and has a redder complexion when cold or hot, and emotional excitement. Severe cases also form a sedimentary stain, which is difficult to cure.

Collagen is a protein widely distributed in human connective tissue, and is the most abundant protein in human body, accounting for 25% to 35% of the total protein. Its main functions are in the maintenance of extracellular environment, the maintenance of normal physiological functions of tissue and organ, the repair of body damage, etc. The collagen is a natural biological resource, has incomparable excellent biological histocompatibility, supporting elasticity and degradability for other high molecular materials. Therefore, the collagen can be widely applied to industries such as medicine, cosmetics and the like.

However, natural collagen is insoluble in water, has inhomogeneous properties, is difficult to be utilized by human body, and often needs to be treated by chemical means before being used. In addition, the collagen products sold in the market at present are all taken from animal tissues such as pigs, cows, fish and the like, are difficult to avoid virus infection, are not compatible with human bodies, and can cause immunological rejection and allergic symptoms. Therefore, the current collagen can only be used in cosmetics and health care products, and cannot play the original biological function of the collagen at all.

To solve the problems of traditional animal collagens, many scholars have begun to apply biotechnology to produce recombinant collagens. The production of collagen by recombinant microorganisms has become the mainstream due to the convenience of microbial culture. Van generation is closed, et al use Escherichia coli high density fermentation culture production recombinant human collagen, and has been successfully applied to the cosmetic field, has realized the industrial production (Chinese patent 201310157411.7, 201510883010.9 etc.). However, the bacterial expression system has biological safety problems of endotoxin, pyrogen and the like, so that the production and detection cost of the product is high, and potential safety hazards exist; the expressed protein exists in bacterial cells in the form of inclusion bodies, the purification of products is difficult, and the recovery rate is limited; in addition, the prokaryotic expression system is lower in grade, and can not finish post-translational processing modification of an expression product, so that the product has no biological activity.

Therefore, more and more studies have begun to produce recombinant collagen using eukaryotic microorganisms. Yeasts have natural advantages. Firstly, the yeast has a long history of use in the industries of food, pharmaceutical chemicals and the like, can be used for secretion and expression of exogenous protein, is beneficial to a downstream separation and purification process, and does not have the biosafety problems of endotoxin, heat sources and the like bacteria.

Disclosure of Invention

The invention aims to provide a recombinant human-derived collagen with excellent hydrophilicity, a pichia pastoris engineering bacterium for secreting and expressing the recombinant human-derived IxIII collagen and application thereof, and can efficiently and safely carry out extracellular secretory expression of the recombinant human-derived IxIII collagen.

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

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

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

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

In a specific embodiment, the invention 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, and the amino acid sequence is shown in SEQ NO. 5.

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

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

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

The constructed strain secreting and expressing the recombinant human IxIII type collagen IxIII 495aa is Pichia pastoris JY0501, the preservation number is CGMCC No.16464, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 2018, 9 and 12 days, and the preservation address is No.3 of the national institute of sciences, No.3 of West Lu 1 of the sunward area in Beijing. The Pichia pastoris JY0501 is constructed by inducing a linear recombinant human IxIII collagen plasmid ppic9K-IxIII obtained by Sac I enzyme digestion into the Pichia pastoris.

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

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

The invention selects nucleotide sequences with strong water solubility and stability from the I-type and III-type helical regions of human collagen genes with known sequences, inserts optimized gene segments into pichia pastoris expression plasmids, converts pichia pastoris to screen high-expression pichia pastoris gene engineering bacteria, and obtains high-purity recombinant human-like collagen through preliminary fermentation and purification steps. The recombinant human ppic9K collagen has good hydrophilicity and stability, has 100 percent of same structure with the corresponding part of a natural collagen gene sequence, can not cause immunological rejection when being applied to a human body, and has potential application prospect in the fields of biomedical materials, cosmetics and the like.

Drawings

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

FIG. 2 is a chart showing the hydrophobicity analysis of amino acids in human type III alpha 1 chain collagen.

FIG. 3 is a diagram of hydrophobicity analysis of IxIII 495aa amino acid 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 shows the Sac I agarose gel electrophoresis of recombinant human IxIII collagen plasmid ppic 9K-IxIII.

FIG. 6 is a photograph of a Pichia pastoris GS115 after electroporation.

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

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

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

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

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

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

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

Fig. 14 is a graph showing the effect results.

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

Fig. 16 is a graph of texture improvement rate.

Fig. 17 is a view of the front and rear faces of A, B, C test subjects.

Detailed Description

The present invention will be described in more detail with reference to the following examples and the accompanying drawings. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1

1. Protein sequence selection

The results of the hydrophobicity analyses of the amino acid sequence of human type I alpha 1 chain collagen (SEQ No.1) and the amino acid sequence of human type III alpha 1 chain collagen (SEQ No.2) are shown in FIGS. 1 and 2. The lower the hydrophobicity evaluation score, the better the hydrophilicity. According to the results of hydrophobicity analysis, selecting low-scoring amino acid fragments, taking 69 amino acid short peptide fragments of type I collagen (namely, recombinant human source type I collagen I69 aa, SEQ No.3) and 96 amino acid short peptide fragments of human type III collagen (recombinant human source type III collagen III 96aa, SEQ No.4), connecting the two short peptide fragments into a 165 short peptide fragment unit (namely, recombinant human source IxIII collagen IxIII 165aa, SEQ No.5), connecting and repeating the fragment unit head and tail for three times to integrate into new protein, namely, the recombinant human source IxIII collagen (namely, recombinant human source IxIII collagen IxIII 495aa, SEQ No. 6). The results of the hydrophobicity analysis of the amino acids of the recombinant human IxIII type collagen are shown in FIG. 3, and the hydrophobicity evaluation of all the amino acids in the protein is lower than zero, which indicates that the hydrophilicity of the protein is good.

2. Plasmid construction and linearization

The recombinant human IxIII type collagen IxIII 495aa is translated into a base sequence (SEQ No.7), a PAS (PCR-based Accurate Synthesis) method is adopted to synthesize a gene, the gene is double-digested and connected between Xho I and Not I of IxIII 495aa to ppic9K carriers, and FIG. 4 is a schematic construction diagram of a recombinant human IxIII type collagen plasmid ppi 9K-IxIII. The obtained recombinant plasmid ppic9K-IxIII is transferred into a TOP10 clone strain, positive clones are picked for sequencing, and the sequencing result is shown as SEQ No. 8. The region at bases 244-249 and 1756-1763 of SEQ ID No.8 is the site for cleavage.

Extracting 20 mu g of plasmid, using Sac I to cut linearization, freezing and concentrating for use. Digest for 3h at 37 ℃. mu.L of the sample was subjected to 1% agarose gel electrophoresis, and the results of the electrophoresis are shown in FIG. 5. Wherein M is a DNA standard substance, and is 1000, 2000, 3000, 4000, 5000, 6000, 8000 and 10000bp from bottom to top; 1 is Sac I enzyme digestion; 2 is the recovered target fragment.

TABLE 1 preparation table of enzyme digestion linearization system

3. Pichia electrotransformation cell GS115

The electric rotor was ice-cooled, 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 rotor having a diameter of 0.2cm, and the electric rotor was ice-cooled for 5 min. The electric shock conditions are as follows: the voltage is 1.5 kV; a capacitance of 25 μ F; the resistance is 200 omega, and the electric shock time is 4-10 msec. After the electric shock was completed, 650uL of sorbitol solution with a pre-cooled concentration of 1M on ice was added to the electric shock conversion cup, and the solution was gently and uniformly blown with a pipette tip. The whole liquid in the cuvette was transferred to a new 2ml EP tube and incubated at 30 ℃ for 2 hours. And (4) carrying out low-speed centrifugation to collect thalli, coating all thalli on an MD (MD) plate, and culturing at constant temperature of 30 ℃ for 3-4 days. FIG. 5 is a photograph of a Pichia pastoris GS115 after electroporation.

PCR identification of Positive clones

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

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

5. Small test expression

Inducing expression: taking 50 μ L of the identified positive strain (No. 5, No.6, No.7, No. 8), inoculating into a conical flask containing 10ml of BMGY, culturing overnight at 30 ℃ at 220r/min, and shaking until OD600 is 2-6 (logarithmic growth, about 16-18 h); centrifuging at room temperature for 5min at 5000r/min, collecting cells, removing supernatant, resuspending the cells with 10ml BMMY, and performing induced expression; sampling 1mL of the culture medium every 24h, and adding methanol to a final concentration of 0.5% to continue induction; centrifuging the sample at 10000r/min for 2min at the following time points of 0, 24, 48, 72 and 96hr, and collecting the supernatant for detection.

And (3) concentrating the expression product by a trichloroacetic acid precipitation method:

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

(2) centrifuging at 12000r/min for 10min to obtain viscous yellowish brown jelly, removing supernatant, collecting precipitate, placing the EP tube on absorbent paper, and standing in a 37 ℃ oven for 10-20 min to ensure that no obvious liquid remains on the tube wall;

(3) adding 200 mu L of cold acetone, oscillating and uniformly mixing, standing the sample at room temperature for 10min, and washing off residual TCA on the tube wall and the tube bottom;

(4) centrifuging at 12000r/min for 10min, discarding the supernatant, repeating the steps (2) and (3), and repeating 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 does not dissolve, the precipitate can be blown up with a 100. mu.L lance tip until the precipitate dissolves.

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

8# positive bacteria are taken for strain preservation, named as Pichia pastoris JY0501, and the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (the preservation center address: Beijing city rising district Beijing West Lu No.1 Hopkin, institute of microbiology, China academy of sciences, postal code: 100101) in 9, 12 and 2018, and the preservation number is CGMCC No. 16464.

The invention selects nucleotide sequences with good water solubility and strong stability from the I-type and III-type helical regions of human collagen genes with known sequences, inserts optimized gene segments into pichia pastoris expression plasmids, converts pichia pastoris to screen high-expression pichia pastoris gene engineering bacteria, and obtains high-purity recombinant human-like 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 the corresponding part of a natural collagen gene sequence, so that the recombinant human-like collagen can not cause immunological rejection when being applied to a human body, and can be widely applied to the fields of biomedical materials, cosmetics and the like. The secretory expression vector is adopted, the secretory expression of the recombinant human-like collagen is successfully realized, the expression product is secreted in the supernatant, the purification is convenient, the advantages which are not possessed by other recombinant human-like collagen production are realized, and the large-scale production operation is convenient. After the carrier is electrically transferred into the pichia pastoris, the gene is integrated on a pichia pastoris genome, so the recombinant strain has good stability, the gene is not easy to lose after multiple passages, the character of high-efficiency expression can be kept, stable production can be well realized, the pichia pastoris production method is aerobic fermentation, the thallus density is high, and the expression amount has a great promotion space.

Example 2

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

2. Effect determination

Effect ═ 100% pre-treatment skin loss count-post-treatment skin loss count/pre-treatment skin loss count.

Effect level:

the effect is shown: the red swelling or stabbing pain is reduced by 60 to 90 percent, and the symptoms are relieved;

the method has the following advantages: the red swelling or stabbing pain is reduced by 20 to 60 percent, and the symptoms are improved;

and (4) invalidation: the red swelling or stabbing pain subsided by less than 20%, and the clinical symptoms were not improved.

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

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

TABLE 2A group 0.1% recombinant human IxIII collagen IxIII 495aa test results

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

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

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

-texture: each time point was statistically significant.

TABLE 3B group 0.3% recombinant human IxIII collagen IxIII 495aa test results

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

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

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

-texture: has statistical significance.

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

TABLE 4C group 0.5% recombinant human IxIII collagen IxIII 495aa test results

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

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

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

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

To sum up, A, B, C three groups of comparison, through continuous use of a 15-day test person, 4 times of analysis on four aspects of moisture, effect, red blood silk surface area and texture, the detection result shows that the recombinant human IxIII collagen IxIII 495aa with the concentration of 0.2-0.4% is mild and non-irritating, the problem of the facial red blood silk can be effectively relieved, and the effect of relieving and repairing is achieved.

Sequence listing

<110> Jiangsu Jiangshan Convergence Biotech Co., Ltd

<120> recombinant human IxIII collagen, expression strain and application thereof

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

740 745 750

Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro

755 760 765

Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro

915 920 925

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

930 935 940

Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val

945 950 955 960

Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro

965 970 975

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

980 985 990

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

995 1000 1005

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

1010 1015 1020

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

1025 1030 1035 1040

Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro

1045 1050 1055

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

1060 1065 1070

Gly Pro Ala Gly Pro Val Gly Pro Val Gly Ala Arg Gly Pro Ala Gly

1075 1080 1085

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

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

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

1140 1145 1150

Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg

1155 1160 1165

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

1170 1175 1180

Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe

1185 1190 1195 1200

Leu Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr

1205 1210 1215

Arg Ala Asp Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp

1220 1225 1230

Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro

1235 1240 1245

Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met

1250 1255 1260

Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln

1265 1270 1275 1280

Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly

1285 1290 1295

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