阅读说明：本技术 重组人源Ⅰ型胶原蛋白Ⅰα1x6、表达菌株及其应用 (Recombinant human type I collagen I alpha 1x6, expression strain and application thereof ) 是由 赵健烽 朱逸丽 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Ⅰ型胶原蛋白Ⅰα1x6、表达菌株及其应用。所述的重组人源Ⅰ型胶原蛋白Iα1xN由若干个重组人源Ⅰ型胶原蛋白I 69aa亲水性片段串联而成。本发明的重组人源Ⅰ型胶原蛋白表达菌株能够有效稳定和大量地表达重组人源Ⅰ型胶原蛋白。本发明的重组人源胶原蛋白具有良好的亲水性及稳定性,其69氨基酸长度的重复短肽结构与天然胶原蛋白基因序列相应部分100％相同,应用于人体中不会造成免疫排斥。本发明的重组人源Ⅰ型胶原蛋白与传统的罗非鱼皮胶原相比,能够显著提高皮肤含水量、改善皱纹尺寸、深度和纹理,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 type I collagen I alpha 1x6, an expression strain and application thereof. The recombinant human I-type collagen I alpha 1xN is formed by connecting a plurality of recombinant human I-type collagen I69 aa hydrophilic segments in series. The recombinant humanized type I collagen expression strain can effectively and stably express the recombinant humanized type I collagen in large quantity. The recombinant human collagen has good hydrophilicity and stability, the repeated short peptide structure of 69 amino acids is 100% identical to the corresponding part of the natural collagen gene sequence, and the recombinant human collagen can not cause immunological rejection when being applied to a human body. Compared with the traditional tilapia skin collagen, the recombinant human type I collagen of the invention can obviously improve the water content of skin, the size, the depth and the texture of wrinkles, and can be widely applied to the fields of biomedical materials, cosmetics and the like.)

1. The amino acid sequence of the recombinant human I-type collagen I69 aa is shown in SEQ No. 2.

2. The recombinant humanized I-type collagen I alpha 1xN is formed by connecting N sections of recombinant humanized I-type collagen I69 aa shown in SEQ No.2 in series, wherein N is more than or equal to 2.

3. The recombinant human type I collagen I alpha 1xN of claim 2, which is recombinant human type I collagen I alpha 1x6, and is formed by connecting 6 segments of recombinant human type I collagen I69 aa shown in SEQ No.2 in series, and the amino acid sequence is shown in SEQ No. 3.

4. The coding gene of the recombinant human type I collagen I alpha 1x6 of claim 3, the nucleotide sequence is shown in SEQ No. 4.

5. The recombinant human type I collagen plasmid ppic9K-I alpha 1x6 has a nucleotide sequence shown in SEQ No. 5.

6. The method for constructing the recombinant human type I collagen plasmid ppic 9K-lalpha 1x6 as claimed in claim 5, wherein the recombinant human type I collagen plasmid is constructed by double-digesting and ligating the coding gene of the recombinant human type I collagen lalpha 1x6 between Xho I and Not I of ppic9K vector.

7. The recombinant human type I collagen I alpha 1x6 expression strain is Pichia pastoris JY0401 with the preservation number of CGMCC No. 16463.

8. The use of the recombinant human type I collagen I α 1x6 according to claim 3 in the preparation of wrinkle-removing skin care products.

9. The use according to claim 8, wherein the concentration of the recombinant human type I collagen I alpha 1x6 in the wrinkle-removing skin care product is 0.1-0.75% w/v.

10. The use according to claim 9, wherein the wrinkle-removing skin care product contains recombinant human type I collagen I α 1x6 at a concentration of 0.5-0.75% w/v.

Technical Field

The invention belongs to the technical field of bioengineering, and relates to a recombinant humanized type I collagen I alpha 1x6, a pichia pastoris engineering bacterium for expressing the recombinant humanized type I collagen I alpha 1x6 and application thereof.

Background

Collagen is a biopolymer substance, and is an important substance for maintaining the morphology and structure of skin and tissues and organs and repairing various damaged tissues. It is mainly present in the skin, bone, cartilage, teeth, tendons, ligaments and blood vessels of humans and animals, and is an important structural protein in connective tissues. The collagen can provide necessary nutrients for skin layers containing the collagen, so that the activity of the collagen in the skin is enhanced, the integrity of stratum corneum moisture and a fiber structure is maintained, the living environment of skin cells is improved, the metabolism of skin tissues is promoted, the circulation is increased, and the aims of moistening the skin, delaying aging, beautifying, removing wrinkles and nourishing hair are fulfilled.

The conventional method for producing collagen is to treat animal tissues (pigskin, cow skin, donkey skin, fish skin/scale, etc.) with acid, alkali or enzyme to extract collagen therefrom. Although these methods are low in cost and high in recovery rate, they have the following problems: (1) the prepared collagen is a mixed collagen peptide segment with small molecular weight and unequal length, is commonly called gelatin, and has difficult effects of moisturizing and nourishing; (2) the extraction process is simple and extensive, the product purity is not good, and peculiar smell is common; (3) the collagen is derived from animals, so that the potential biological safety hazard cannot be eliminated, and the wide application of the traditional collagen in various products such as cosmetics, foods, medicines and the like is greatly limited; (4) a great amount of waste water generated during processing and extraction seriously harms the environment; (5) the technology threshold is low, the raw material source is not easy to control, the leather shoe milk and poison capsules move transversely, and the potential safety hazard of food and medicine is serious.

To solve a series of problems of the conventional animal collagen, many scholars begin to apply biotechnology to produce recombinant collagen. 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 costs of the product are high, and hidden dangers 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 scholars are beginning 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 type I collagen with excellent hydrophilicity, a pichia pastoris engineering bacterium for secretory expression of the recombinant human type I collagen and application thereof, and the recombinant human type I collagen can be efficiently and safely expressed in an extracellular secretory manner.

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. 2.

The recombinant humanized I-type collagen I alpha 1xN is formed by connecting N sections of recombinant humanized I-type collagen I69 aa shown in SEQ No.2 in series, wherein N is more than or equal to 2.

In a specific embodiment, the invention provides a recombinant human type I collagen I alpha 1x6, i.e. I414 aa, which is formed by connecting 6 segments of recombinant human type I collagen I69 aa shown in SEQ No.2 in series, and the amino acid sequence is shown in SEQ No. 3. Its theoretical molecular weight is about 38.345 kd.

The nucleotide sequence of the coding gene of the recombinant human type I collagen I alpha 1x6 is shown in SEQ No. 4.

The nucleotide sequence of the recombinant human type I collagen plasmid ppic9K-I alpha 1x6 constructed by the invention is shown in SEQ No.5, and the recombinant human type I collagen plasmid is constructed by connecting the coding gene of the recombinant human type I collagen I alpha 1x6 to EcoR I and Not I of ppic9K vector through double enzyme digestion.

The constructed strain for secreting and expressing the recombinant human type I collagen is Pichia pastoris JY0401, the preservation number is CGMCC No.16463, the strain is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms in 2018, 9 and 12 months, and the preservation address is No.3 of the national institute of sciences, No.1 of Xilu, North Chen of the Yangxi district, Beijing city. The Pichia pastoris JY0401 is constructed by inducing a linearized recombinant human type I collagen plasmid ppic9K-I alpha 1x6 obtained by Sac I enzyme digestion into Pichia pastoris.

The invention also provides application of the recombinant human type I collagen I alpha 1x6 in preparation of wrinkle-removing skin care products.

Preferably, in the wrinkle-removing skin care product, the concentration of the recombinant human type I collagen I alpha 1x6 is 0.1-0.75% (w/v, g/mL), more preferably 0.5-0.75% (w/v, g/mL).

The invention selects a nucleotide sequence with strong water solubility and stability from a helical region of a human collagen gene type I with a known sequence, inserts an optimized gene segment into a pichia pastoris expression plasmid, converts pichia pastoris and screens to obtain a high-expression pichia pastoris gene engineering bacterium, and obtains high-purity recombinant human-like collagen through preliminary fermentation and purification steps. The recombinant human type I collagen has good hydrophilicity and stability, the structure of the recombinant human type I collagen is 100 percent identical to the corresponding part of a natural collagen gene sequence, and immunological rejection can not be caused when the recombinant human type I collagen is applied to a human body. Compared with the traditional tilapia skin collagen, the recombinant human type I collagen of the invention can obviously improve the water content of skin, the size, the depth and the texture of wrinkles, 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 graph showing the hydrophobicity analysis of amino acids in recombinant human type I collagen.

FIG. 3 is a schematic diagram of the construction of recombinant human type I collagen plasmid ppic 9K-I.alpha.1 x 6.

FIG. 4 shows the electrophoresis of Sc I digested agarose gel of recombinant human type I collagen plasmid ppic 9K-I.alpha.1 x 6.

FIG. 5 is a photograph of a Pichia pastoris GS115 after electroporation.

FIG. 6 is a gel electrophoresis of the positive clone strains.

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

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

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

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

FIG. 11 is a graph showing protein expression analysis of samples cultured for 0, 24, 48, 72 and 96 hours for #10 positive clonal strain.

FIG. 12 is a graph showing the results of the moisture improvement rate.

Fig. 13 is a graph showing the wrinkle improvement rate results.

Fig. 14 is a graph showing the results of texture improvement rates.

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 amino acid sequence (SEQ No.1) of human type I alpha 1 chain collagen was subjected to hydrophobicity analysis, and the results are shown in FIG. 1. The lower the hydrophobicity evaluation score, the better the hydrophilicity. According to the result of hydrophobicity analysis, selecting amino acid fragments with low scores, taking 69 short peptide unit fragments (namely recombinant human type I collagen I69 aa and SEQ No.2), and connecting and repeating the ending of the short peptide fragments for six times to integrate the short peptide fragments into new protein, namely the recombinant human type I collagen (namely recombinant human type I collagen I alpha 1x6 and SEQ No. 3). The results of the hydrophobicity analysis of the amino acids of the recombinant human type I collagen are shown in figure 2, and the hydrophobicity evaluation of all the amino acids in the protein is lower than zero, which indicates that the protein has good hydrophilicity.

2. Plasmid construction and linearization

The recombinant human type I collagen I alpha 1x6 is translated into a base sequence (SEQ No.4), a PAS (PCR-based Accurate Synthesis) based method is adopted to synthesize a gene I alpha 1x6, the gene I alpha 1x6 is double-digested and connected between Xho I and Not I of ppic9K vector, and FIG. 3 is a construction schematic diagram of a recombinant human type I collagen plasmid ppic9K-I alpha 1x 6. The obtained recombinant plasmid ppic9K-I alpha 1x6 is transferred into a TOP10 clone strain, positive clones are picked for sequencing, and the sequencing result is shown as SEQ No. 5. The region at bases 244-249 and 1513-1521 of SEQ ID No.5 is the site of restriction enzyme.

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. 4. 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.

Screening for high copy clones by G418

The colonies growing on the MD plate were scraped off with a sterile tip, suspended in 1ml of YPD medium, 50. mu.L of the suspension was applied to YPD +0.5mg/ml G418, YPD +1mg/ml G418, YPD +2mg/ml G418, YPD +3mg/ml G418 and YPD +4mg/ml G418 plates, and incubated at 30 ℃ for 3 to 4 days.

PCR identification of Positive cloned Strain

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.

6. Small test expression

Inducing expression: taking 50 μ L of the identified positive strain (7#, 8#, 9#, 10#), inoculating into a conical flask containing 10ml of BMGY, culturing overnight at 30 ℃ and 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.

Expression analysis:

SDS-PAGE electrophoretic detection: the results of expression analysis of 7#, 8#, 9#, 10# positive bacteria are shown in fig. 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 JY0401, and the strain is preserved in the common microorganism center of China Committee for culture preservation of microorganisms in 2018, 9, 12 and 7 (the preservation center address is No.3 of Beijing Kogyo Chen-Xilu No.1 of Beijing, institute of microbiology, China academy of sciences, postal code is 100101), and the preservation number is CGMCC No. 16463.

The invention selects a nucleotide sequence with good water solubility and strong stability from a helical region of human collagen gene type I with a known sequence, inserts an optimized gene segment into a pichia pastoris expression plasmid, converts pichia pastoris and screens to obtain 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

For the control group (comparative example 1, tilapia skin collagen group) and the effect group (recombinant human type I collagen type I alpha 1x6), Chinese women aged 35-55 years old and with fine lines and wrinkles on the face were selected, and 30 persons were each treated with one bottle at night every day. The test was performed in a double blind test, with the four indicators of moisture, overall wrinkle size, wrinkle depth and texture on each panelist's face at week 0 and 8 using a Milipure Intelligent skin moisture tester and an Antera3D skin tester, respectively.

(1) Control group (comparative example 1)

The components: tilapia skin collagen, concentration 0.75% (w/v, g/mL, i.e. 7.5g/L)

Weighing tilapia skin collagen powder, fully stirring and dissolving, sterilizing and filtering at 0.22 mu m in a hundred-grade area under a ten-thousand-grade background, subpackaging and freeze-drying, wherein the amount of each part of freeze-dried tilapia skin collagen powder is 15mg of solid. The solvent used was 2ml of purified water.

(2) The components: recombinant human type I collagen at a concentration of 0.075% (w/v, g/mL, i.e. 0.75g/L)

Weighing recombinant human type I collagen with the purity of 99%, fully stirring and dissolving, performing aseptic filtration by a 0.22 mu m filter membrane according to GMP requirements, packaging and freeze-drying in ten thousand grades, wherein the solid content of each part is 1.5mg after freeze-drying. The solvent used was 2ml of purified water.

(3) The components: recombinant human type I collagen at a concentration of 0.1% (w/v, g/mL, i.e., 1g/L)

Weighing recombinant human type I collagen with the purity of 99%, fully stirring and dissolving, performing aseptic filtration by a 0.22 mu m filter membrane according to GMP requirements, packaging and freeze-drying in ten thousand grades, and filling 2mg of solid in each bottle after freeze-drying. The solvent used was 2ml of purified water.

(4) The components: recombinant human type I collagen at a concentration of 0.25% (w/v, g/mL, i.e., 2.5g/L)

Weighing recombinant human type I collagen with the purity of 99%, fully stirring and dissolving, performing aseptic filtration by a 0.22 mu m filter membrane according to GMP requirements, packaging and freeze-drying in ten thousand grades, and filling 5mg of solid in each bottle after freeze-drying. The solvent used was 2ml of purified water.

(5) The components: recombinant human type I collagen at a concentration of 0.5% (w/v, g/mL, i.e., 5g/L)

Weighing recombinant human type I collagen with the purity of 99%, fully stirring and dissolving, performing aseptic filtration by a 0.22 mu m filter membrane according to GMP requirements, packaging and freeze-drying in ten thousand grades, and filling 10mg of solid in each bottle after freeze-drying. The solvent used was 2ml of purified water.

(6) The components: recombinant human type I collagen at a concentration of 0.75% (w/v, g/mL, i.e., 7.5g/L)

Weighing recombinant human type I collagen with the purity of 99%, fully stirring and dissolving, performing aseptic filtration by a 0.22 mu m filter membrane according to GMP requirements, packaging and freeze-drying in ten thousand grades, and filling 15mg of solid in each bottle after freeze-drying. The solvent used was 2ml of purified water.

Tables 2-7 show the results of the facial skin index tests of the experimenters using the tilapia skin collagen of 0.75% w/v of the control group and the recombinant human type I collagen of different concentrations before and after 8 weeks of use. Fig. 12 is a graph showing the results of the moisture improvement rate, fig. 13 is a graph showing the results of the wrinkle improvement rate, and fig. 14 is a graph showing the results of the texture improvement rate. From tables 2-7 and fig. 12-14, it can be seen that the recombinant human type i collagen is applied to human skin, the texture is shallower than that before use, the moisture content of the skin is correspondingly increased, the overall size of wrinkles and the wrinkle depth are both obviously improved, and compared with the traditional tilapia skin collagen, the wrinkle removing effect is obvious.

TABLE 2 test results for 0.75% w/v tilapia skin collagen in control group

TABLE 3 test results for effect group concentration 0.075% w/v recombinant human type I collagen

TABLE 4 test results for recombinant human type I collagen at an effect group concentration of 0.1% w/v

TABLE 5 test results for recombinant human type I collagen at an effect group concentration of 0.25% w/v

TABLE 6 test results for recombinant human type I collagen at effect panel concentration of 0.5% w/v

TABLE 7 test results for Effect group concentration 0.75% w/v recombinant human type I collagen

Sequence listing

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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> 69

<212> PRT

<213> Homo sapiens

<400> 2

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> 3

<211> 414

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410

<210> 4

<211> 1242

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggtccacaag gtccaagagg tgataagggt gaaactggtg aacaaggtga cagaggtatc 60

aagggtcaca gaggtttctc tggattgcaa ggtccacctg gtccaccagg ttctccaggt 120

gagcaaggtc cttctggtgc ttctggtcct gctggaccaa gaggtcctcc aggatctgct 180

ggtgctccag gtaaagatgg tttgaacggt cctcaaggac ctcgtggtga caaaggtgaa 240

acaggcgagc agggtgatcg tggtattaag ggacatagag gattttccgg tctgcaggga 300

cctccaggtc ctcctggtag tccaggtgaa cagggaccaa gtggtgctag tggacctgcc 360

ggtcctagag gcccacctgg ttcagctggt gcacctggaa aggatggtct taatggacca 420

cagggaccta gaggcgacaa gggcgagaca ggcgaacaag gcgatagggg aatcaaaggt 480

cataggggtt ttagcggact tcagggacca ccaggaccac ctggatctcc cggcgaacag 540

ggtccatcag gtgcttcagg cccagctggt cccaggggac ctcctggttc tgcaggcgcc 600

cctggtaaag acggacttaa tggtcctcag ggtccacgtg gcgataaggg cgaaaccggt 660

gagcagggcg atagaggcat taagggtcat cgtggattca gtggattaca aggccctcca 720

ggacctccag gctcaccagg tgaacaaggc ccatccggtg caagtggtcc agctggccct 780

cgtggcccac caggatcagc aggcgctccc ggcaaggacg gtttaaatgg acctcaaggc 840

ccaaggggag acaaaggcga gactggtgag caaggcgacc gtggcatcaa aggacaccgt 900

ggattttctg gcttacaggg ccctcctggt ccacctggaa gccctggcga acaaggacct 960

tcaggtgcat ctggaccagc aggccccaga ggaccaccag gtagtgctgg cgctcctgga 1020

aaagatggcc taaacggacc ccaaggtcct agaggtgaca aaggcgaaac aggtgaacag 1080

ggcgacagag gaatcaaagg acatcgtggt ttctccggtc tacaaggtcc tcctggacct 1140

cctggtagcc ccggtgagca aggccccagt ggtgcttcag gtcccgcagg tcctcgtgga 1200

ccaccaggtt ccgctggcgc accaggtaag gacggattga at 1242

<210> 5

<211> 1520

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

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 tgaagctggt ccacaaggtc caagaggtga taagggtgaa 300

actggtgaac aaggtgacag aggtatcaag ggtcacagag gtttctctgg attgcaaggt 360

ccacctggtc caccaggttc tccaggtgag caaggtcctt ctggtgcttc tggtcctgct 420

ggaccaagag gtcctccagg atctgctggt gctccaggta aagatggttt gaacggtcct 480

caaggacctc gtggtgacaa aggtgaaaca ggcgagcagg gtgatcgtgg tattaaggga 540

catagaggat tttccggtct gcagggacct ccaggtcctc ctggtagtcc aggtgaacag 600

ggaccaagtg gtgctagtgg acctgccggt cctagaggcc cacctggttc agctggtgca 660

cctggaaagg atggtcttaa tggaccacag ggacctagag gcgacaaggg cgagacaggc 720

gaacaaggcg ataggggaat caaaggtcat aggggtttta gcggacttca gggaccacca 780

ggaccacctg gatctcccgg cgaacagggt ccatcaggtg cttcaggccc agctggtccc 840

aggggacctc ctggttctgc aggcgcccct ggtaaagacg gacttaatgg tcctcagggt 900

ccacgtggcg ataagggcga aaccggtgag cagggcgata gaggcattaa gggtcatcgt 960

ggattcagtg gattacaagg ccctccagga cctccaggct caccaggtga acaaggccca 1020

tccggtgcaa gtggtccagc tggccctcgt ggcccaccag gatcagcagg cgctcccggc 1080

aaggacggtt taaatggacc tcaaggccca aggggagaca aaggcgagac tggtgagcaa 1140

ggcgaccgtg gcatcaaagg acaccgtgga ttttctggct tacagggccc tcctggtcca 1200

cctggaagcc ctggcgaaca aggaccttca ggtgcatctg gaccagcagg ccccagagga 1260

ccaccaggta gtgctggcgc tcctggaaaa gatggcctaa acggacccca aggtcctaga 1320

ggtgacaaag gcgaaacagg tgaacagggc gacagaggaa tcaaaggaca tcgtggtttc 1380

tccggtctac aaggtcctcc tggacctcct ggtagccccg gtgagcaagg ccccagtggt 1440

gcttcaggtc ccgcaggtcc tcgtggacca ccaggttccg ctggcgcacc aggtaaggac 1500

ggattgaatt aagcggccgc 1520