CN112210003A - Crystal structure and application of recombinant apolipoprotein J and analogue thereof - Google Patents

Crystal structure and application of recombinant apolipoprotein J and analogue thereof Download PDF

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CN112210003A
CN112210003A CN202010644415.8A CN202010644415A CN112210003A CN 112210003 A CN112210003 A CN 112210003A CN 202010644415 A CN202010644415 A CN 202010644415A CN 112210003 A CN112210003 A CN 112210003A
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宇文镐
陈翔
杜佩云
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Dexinwonder Medical Technology Co ltd
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Abstract

The invention provides a crystal structure of a recombinant apolipoprotein J and an analogue thereof and application thereof, wherein the primary sequence of amino acid is any one of SEQ ID NO. 1-28. The spatial structure of the recombinant apolipoprotein J has no artificial addition structure, is not easy to induce immunogenic reaction, has no other toxic or side effect, and has strong druggability; meanwhile, the purified product has obvious immunological activity, biological activity and drug effect, can effectively treat diseases of tested animals, and has good application value.

Description

Crystal structure and application of recombinant apolipoprotein J and analogue thereof
Technical Field
The invention relates to the technical field of biology, and particularly relates to a recombinant apolipoprotein J, a preparation method and application thereof.
Background
Apolipoprotein was originally isolated from the testis network fluid of ram, and since it promotes the agglutination of Sertoli cells and supports the maturation and development of sperm, it was also named as agglutination factor, widely expressed in tissues and closely related to various physiological and pathological processes, including Apolipoprotein A-I, B, E, J. Up to now, the functions of apolipoproteins have been reported to include the inhibition of apoptosis (Kowolk et al 2006), the inactivation of complement factors (Correa-Rotter et al 1992), the recovery and transport of lipids (Gelissen et al 1998), the protection of cell membranes, and the maintenance of intercellular or cellular-matrix associations, among others.
Apolipoprotein J (Apo J, also known as Clusterin) is a multifunctional thiolated glycoprotein heterodimer containing two 40KD chains and linked by disulfide bonds, the structure of which is associated with the Lipocalin family. Almost all cell types secrete this protein and the human ApoJ gene is widely found in human tissues, with high mRNA levels especially in the brain, adrenal medulla, anterior and posterior pituitary, ovary, testis, and liver. Apo J is involved in many physiological functions including apoptosis, complement regulation, protection of cell membranes, stability of cell-to-cell and cell-to-matrix interactions, DNA repair and carcinogenesis.
A large number of studies at home and abroad show that the apolipoprotein J can be used as a medicament for clinical treatment of various diseases. Although apolipoprotein J is widely present in the human body at present, the source of the natural apolipoprotein J is extremely limited; recombinant apolipoprotein J, which has been commercialized at present, carries no exception to TAGs (TAGs), and these tagged proteins cannot be used in pharmaceuticals. It is necessary to develop a recombinant apolipoprotein J with the same efficacy, which can be used for pharmacy and is convenient for large-scale production.
Disclosure of Invention
In view of the above, the present invention aims to provide a recombinant apolipoprotein J, a preparation method and an application thereof, which aims to achieve the purpose of treating diseases by using the existing apolipoprotein J in the prior art that the yield is low and the apolipoprotein J is not suitable for industrial production.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a recombinant apolipoprotein J, wherein the amino acid primary sequence of the recombinant apolipoprotein J is any one of SEQ ID NO. 1-28.
Further, the recombinant apolipoprotein J consists of an A fragment and a B fragment, wherein the amino acid primary sequence of the A fragment is any one of SEQ ID NO.29-36, and the amino acid primary sequence of the B fragment is any one of SEQ ID NO. 37-38. The A, B segments can be arranged and combined to achieve the same effect. The A, B fragment can be inserted into one expression vector at the same time, or A and B fragments can be inserted into the same two expression vectors respectively, or A and B fragments can be inserted into different two expression vectors respectively, and the fusibility expression or inclusion body expression or secretion expression can be carried out in vitro in the same host.
Further, the A segment and the B segment are connected by one or more of hydrogen bond, disulfide bond and peptide bond.
Still further, the spatial structure of the recombinant apolipoprotein J is shown in figures 1-3, and comprises 2-4 domains, 2-5 beta folds and 2-5 alpha helices.
Further, the molecular structure of the crystal of the recombinant apolipoprotein J comprises 3 structural domains, 3 beta folds and 2 alpha helices
The invention also provides a preparation method of the recombinant apolipoprotein J, which comprises the steps of thallus crushing, inclusion body washing and dissolving, anion chromatography, desalting and liquid changing, cation chromatography, renaturation and SEC chromatography. After the sequence is expressed by a host, the recombinant apolipoprotein J can be directly extracted from a culture solution or a crushed cell supernatant; purifying the recombinant apolipoprotein J by ion exchange column chromatography, affinity column chromatography and molecular exclusion column chromatography after protein precipitation and fractionation according to specific optimal procedures and parameters including pH selection, buffer solution selection and concentration, and additive selection (combination) and concentration; regulating the polymerization of the recombinant apolipoprotein J by adopting a surfactant, a pH regulator, a hydrophobic additive and an antioxidant; desalting by ultrafiltration, dialysis and column chromatography. The separation and purification method comprises one or more of isoelectric point precipitation, ammonium sulfate precipitation/fractionation, protein denaturation, protein renaturation, ion exchange column chromatography, hydrophobic column chromatography, affinity column chromatography, size exclusion gel column chromatography, ultrafiltration and dialysis.
The separation and purification method comprises the following steps: crushing thallus; washing the inclusion body; anion exchange chromatography; cation exchange chromatography; renaturation and ultrafiltration; and (5) purifying the molecular sieve. Preferably, the ultrafiltration is TFF; the molecular sieve purification is SEC chromatography. It should be noted that, in the present invention, the APO-J protein formed by the amino acid sequences shown in SEQ ID Nos. 1-28 or 40-41, and the APO-J protein formed by SEQ ID Nos. 29-36 and 38 can be purified by the above-mentioned method.
The renaturation method comprises one or more of dilution renaturation, dialysis renaturation, column chromatography renaturation and other solid phase adsorption renaturation. The filler adopted by the column chromatography method comprises one or more of apolipoprotein J antibody coupling, heparin coupling, activated sulfydryl coupling filler and metal chelating filler. The column chromatography method comprises specific step gradient, continuous gradient and multiple gradient elution.
Preferably, the renaturation adopts stage dilution, and a certain concentration of detergent is added while introducing the small molecular chaperone and the redox couple into renaturation buffer. In the renaturation process, detergents such as SDS (sodium dodecyl benzene sulfonate), SKL (sodium dodecyl sarcosinate), Triton100, Tween 20 and the like are firstly used for processing, and then effective column chromatography separation is carried out, such as ion exchange column chromatography separation or molecular sieve column chromatography separation, so as to obtain an effective monomer of the recombinant apolipoprotein J; the detergent is then removed by ultrafiltration, dialysis, G25 molecular sieve column chromatography, etc. If the apolipoprotein J is expressed in the form of inclusion bodies, the inclusion bodies are thawed and performed in a specific optimal procedure and parameters, including the choice of pH, the choice and concentration of buffer, the choice (combination) and concentration of additives. For the renaturation mode, the first choice is column renaturation and solid phase adsorption renaturation, and dilution and dialysis renaturation can also be adopted; purifying the denatured protein by column chromatography after denaturation; the small molecular chaperone is adopted to help protein renaturation; the small molecular chaperone can be sodium citrate, arginine, glutathione (reduced form and non-reduced form), cysteine, glycine, SKL (sodium dodecyl sarcosinate), Tween 20 and the like, or a combination of the small molecular chaperones, and a certain redox potential is maintained, so that renaturation can be successfully realized. Purifying the renaturation protein by ion exchange column chromatography, affinity chromatography, hydrophobic column chromatography and molecular exclusion column chromatography.
The renaturation of inclusion body after the above-mentioned sequence is expressed by host, must be implemented under the condition of adding protein folding small molecular chaperone. The small molecular chaperone can be sodium citrate, arginine, glutathione (reduced form and non-reduced form), cysteine, glycine, SKL (sodium dodecyl sarcosinate), Tween 20 and the like, or a combination of the small molecular chaperones, and a certain redox potential is maintained, so that renaturation can be successfully realized.
The recombinant apolipoprotein J is applied to the preparation of medicines for preventing and/or treating diseases.
Further, the disease includes cardiovascular disease, amyloidosis, dry eye. The diseases also include senile dementia, alcoholism, male infertility, etc. Wherein the cardiovascular disease comprises atherosclerosis, ischemic heart disease, coronary heart disease, myocardial and cerebral infarction, hyperlipidemia, hypercholesterolemia, hypertension, hyperglycemia, chronic heart failure, ischemic heart failure, coronary heart failure, intravascular plaque deposition, etc. The protein amyloidosis diseases, primary and secondary protein amyloidosis diseases including light chain (AL), heavy chain (AA), serum amyloid (SAA/AA), transthyretin (TTR), and ocular diseases.
Further, the disease is a gene-deficient disease. Patients with the disease include patients with a lower than normal apolipoprotein J, patients with genetic variation in apolipoprotein J, patients with dysfunction of apolipoprotein J, patients with high or normal High Density Lipoprotein (HDL), patients with high or normal Low Density Lipoprotein (LDL), patients with high or normal ApoA, patients with high or normal ApoB, patients with high or normal ApoA, patients with high or normal cholesterol, patients with high or normal ApoA, patients with high or normal ApoB, patients with high or normal cholesterol, patients with high or low density lipoprotein or ApoA, patients with high or normal low density lipoprotein, and/or high or normal ApoB, and/or high or normal cholesterol, and/or high or normal triglyceride, patients with high blood sugar, patients with high cholesterol, patients with hypertension, patients with atherosclerosis, patients with arteriosclerosis and arteriolosclerosis, patients with myocardial infarction symptoms, patients after the onset of myocardial infarction, patients with cerebrovascular infarction symptoms, patients after the onset of cerebrovascular infarction, patients with coronary heart disease or coronary heart disease symptoms, patients with chronic heart failure, patients with heart failure symptoms, patients with senile dementia, patients with alcoholism, patients with clinical diseases caused by low expression levels and/or loss of function of alcohol dehydrogenase and/or acetaldehyde dehydrogenase, and patients with fertility disorders.
Further, the recombinant apolipoprotein J is applied to the preparation of medicines for treating skin diseases and wounds, longevity medicines, health-care medicines and immunity-improving medicines.
The recombinant apolipoprotein J has the action targets in the human body of apolipoprotein A1, apolipoprotein B100, apolipoprotein B58, various in-vivo proteins prone to amyloidosis, a keratoconjunctival mucin layer and an apolipoprotein J-related cell receptor; the clinical administration mode can be intravenous injection, intravenous drip or intramuscular injection; the form of the pharmaceutical preparation can be injection solution or freeze-dried powder injection. The clinical administration dose is 3-40 mg of the protein contained in each needle; the medication interval is one injection every day, every two days, every three days, every four days, every five days, every six days, every seven days, every eight days, every nine days and every ten days of the dosage. When the traditional Chinese medicine composition is used for treating xerophthalmia, the concentration of the drug protein is 0.5-50 micrograms/milliliter, the dosage of the drug is 1-2 drops/eye, 1-4 times/day, and the treatment course is more than 3 days.
Compared with the prior art, the recombinant apolipoprotein J has the following advantages:
the spatial structure of the recombinant apolipoprotein J has no artificial addition structure, is not easy to induce immunogenic reaction, has no other toxic or side effect, and has strong druggability; meanwhile, the purified product has obvious immunological activity, biological activity and drug effect, is higher than similar products obtained by the prior art, can effectively treat diseases of tested animals, and has good application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a three-dimensional structural diagram of a recombinant apolipoprotein J according to the present invention, showing its protein structures at levels 2, 3 and 4;
FIG. 2 is a three-dimensional structural diagram of a recombinant apolipoprotein J according to the present invention, showing surface amino acid residues thereof;
FIG. 3 is a three-dimensional structural diagram of a recombinant apolipoprotein J according to the present invention, showing an amino acid backbone structure thereof;
FIG. 4 is an SEC-HPLC chromatogram of a recombinant apolipoprotein J according to the present invention;
FIG. 5 is a chromatogram of POROS HQ of recombinant apolipoprotein J according to the present invention;
FIG. 6 is a diagram showing the result of SDS-PAGE of POROS HQ of the recombinant apolipoprotein J according to the present invention;
FIG. 7 is a chromatogram of POROS XS of a recombinant apolipoprotein J according to the invention;
FIG. 8 is a diagram showing the result of SDS-PAGE of POROS XS of a recombinant apolipoprotein J according to the present invention;
FIG. 9 is a non-reducing SDS-PAGE analysis of CuCl2 oxidation results for recombinant apolipoprotein J according to the present invention;
fig. 10 is a technical route chart of a toxicological experiment according to an embodiment of the present invention.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
Example 1 extraction of recombinant apolipoprotein J
1.1 disruption of the cells
(1) Fully resuspending each 1g of thallus (including recombinant SEQ ID. NO10 sequence) with 10mL of suspension solution, centrifuging at 8000rpm and 4 ℃ for 15min, and discarding the supernatant; (2) re-suspending the thallus with 10mL/g of suspended bacteria solution, placing the bacterial suspension under a probe of an ultrasonic crusher, and performing power: crushing at 400W for 3s by ultrasonic treatment and at the interval of 6s for 15min in total until the crushing is complete. The construction of the thallus is the prior art and is not described herein again. Preferably, the ratio of the suspended bacteria solution: 50mM Tris-HCl, pH 8.0.
1.2 Inclusion body washes
(1) Centrifuging the crushed thallus at 15000g and 4 ℃ for 30nim, discarding the supernatant, cleaning with a washing solution, adding 10mL of the washing solution per gram of thallus, adding the washing solution, fully suspending, and performing shake incubation at 4 ℃ for 1h, centrifuging at 15000g and 4 ℃ for 30 min; (2) the precipitate obtained by the final centrifugation was dissolved in 10ml/g of a dissolving solution, and then the solution was centrifuged at 15000g and 4 ℃ for 30nim to obtain a supernatant.
As an example of the present invention, 30mg of inclusion bodies can be obtained from 1g of wet bacteria; preferably, the wash solution comprises: washing solution 1:50 mM Tris-HCl, 2M NaCl, 2mM EDTA, pH 8.0; washing solution 2: 50mM Tris-HCl, 1% Triton-100, 2mM EDTA, pH 8.0; washing solution 3: 50mM Tris-HCl, 2M urea, 2mM EDTA, pH 8.0; sequentially cleaning the thallus by using washing solutions 1, 2 and 3; the lysis solution was 50mM Glycine-NaOH, 8M Urea, 10mM DTT, pH 9.0.
1.3 anion exchange chromatography
Column chromatography was performed using POROS HQ, and the column was washed with buffer A (50mM Gly-NaOH, 8M Urea, 10mM DDT, pH9.0), and then with buffer B (50mM Gly-NaOH, 8M Urea, 10mM DDT, 1M NaCl, pH9.0), and collected, and the concentration of the collected sample was 2.7mg/ml, and the yield was 79%, as shown in FIG. 5 and FIG. 6. The specific operation method of the anion exchange chromatography is the prior art, and is not described in detail herein.
1.4 desalting and changing liquid
(1) Connecting the pipeline, the pressure meter and the membrane, rinsing with a buffer solution, exhausting and filling the membrane with the buffer solution; preferably, the flow rate is 200 LMH; (2) putting the inlet into a sample, controlling the transmembrane pressure to be less than 10psi, concentrating until the concentration is 1.5-3 mg/ml, and then changing the solution to 5CV until the pH value of the filtrate is consistent with that of the buffer solution; preferably, the reflux valve of the solution changing (3) is opened after the concentration to 3mg/ml, the flow rate is adjusted to be high, and the air pushes out all the samples, and the results are shown in FIGS. 7 and 8.
1.5 cation exchange chromatography
Column chromatography was performed using POROS XS, washing was performed with buffer A (20mM sodium acetate, 8M urea, 10mM DDT, pH5.5), and then with buffer B (20mM sodium acetate, 8M urea, 10mM DDT, 1M NaCl, pH5.5), and the eluate was collected at a final sample concentration of 1.8mg/ml with a yield of 75%.
1.6 dilution renaturation
15 volumes of buffer 2(10mM PB, 200mM Na-citrate, 5mM GSH, 5mM cysteine, pH7.4, ready for use) were slowly added to the stirred POROS XS eluate to dilute the sample 16-fold, reduce the DTT concentration to 1.25mM, and allowed to stand at 4 ℃ for 4 hours for renaturation. After renaturation, the sample was desalted to buffer 1(10mM PB, pH7.4), concentrated to 0.5mg/ml-1mg/ml, and desalted to buffer 5(10mM PB, 200mM Na-citrate, pH7.4) to prepare a sample.
Preferably, the sample after renaturation can be desalted to buffer solution 1, then concentrated to 3mg-5mg/ml, added with SKL with 3 times of mass and incubated for 16h at 25 ℃ in a stabilization experimental box, and then subjected to Superdex 200; and (3) taking the mobile phase as Buffer 3, and desalting a sample with good purity to Buffer solution 5 to obtain the sample.
As another example of the present invention, 15 volumes of buffer 4(50mM Tris-HCl, 0.5M Arg, 10mM GSH, 1mM GSSG, pH9.0) was slowly added to the stirred POROS XS eluate, the sample was diluted 16-fold, the DTT concentration was reduced to 1.25mM, and the sample was allowed to stand at 4 ℃ for 4 hours for renaturation. And (3) exchanging the TFF solution of the renatured sample into buffer solution 1, concentrating to 5-10mg/ml, adding SKL with the mass of 3 times into the concentrated sample, incubating for 16h in a 25 ℃ stabilization experimental box, and passing through a Superdex 200pg molecular sieve. The mobile phase is Buffer 3, and a sample with good purity is desalted to Buffer5 to prepare a sample.
Preferably, 10 times the molar amount of CuCl is added to the concentrated sample2Standing at normal temperature for 4 hours, adding EDTA with the final concentration of 0.2mM to stop the reaction, and adding SKL to incubate.
1.7 SEC chromatography
The sample after the dilution renaturation is loaded according to 3-5% of the SEC column volume, and is eluted by 10mM PB buffer solution, the parts with the highest purity of the recombinant apolipoprotein J in the eluted samples are combined, and the detection result is shown in figure 4.
Example 2
SEC-HPLC detection was carried out on APO-J (concentration 0.3mg/mL) corresponding to the partial sequence (SEQ ID. NO3, 4, 7, 11, 12, 15, 16, 18, 19, 22, 23, 26, 27) subjected to SEC chromatography, and the results are shown in Table 1. The detection conditions are as follows: TSKGel chromatographic column (300mm is multiplied by 7.8mm), packing particle size is 5 μm; an experimental instrument selects Agilent 1200-DAD GR11010670, a mobile phase is 100mM PBS +200mM urea, and the pH value is 6.7; the sample amount is 80 mul; the flow rate was 0.7 ml/min.
TABLE 1 purity of APO-J corresponding to different sequences by SEC chromatography
Corresponding sequence Purity of the sample% Impurities (a) of Corresponding sequence Purity of the sample% Impurities (a) of
SEQ ID.NO3 94.13 5.86 SEQ ID.NO18 96.12 3.85
SEQ ID.NO4 94.67 5.31 SEQ ID.NO19 95.21 4.76
SEQ ID.NO7 95.11 4.87 SEQ ID.NO22 94.97 5.01
SEQ ID.NO11 93.91 6.05 SEQ ID.NO23 94.38 5.58
SEQ ID.NO12 94.12 5.83 SEQ ID.NO26 93.67 6.29
SEQ ID.NO15 94.86 5.11 SEQ ID.NO27 94.72 5.26
SEQ ID.NO16 95.61 4.35 SEQ ID.NO28 94.31 5.67
It should be noted that, in the present invention, similar results can be obtained by performing SEC-HPLC experiments on the APO-J protein formed by the amino acid sequence shown in SEQ ID Nos. 1-28 and the APO-J protein formed by SEQ ID Nos. 29-36 and 38 by the above method.
Example 3 optimization of the renaturation Process
3.1 Effect of different renaturation Processes on sample stability
The experimental steps are as follows: 6ml of the sample obtained in example 1 after the cation exchange chromatography was taken, and the volume ratio of the sample was adjusted to 1: adding different renaturation buffers into a sample according to a volume ratio of 16, adding the renaturation buffers into the sample, incubating for 4 hours at 4 ℃, directly concentrating or desalting to 20mM PB, concentrating to 3-5mg/ml after pH7.4, incubating for 16 hours with SKL with 3 times of mass, and then passing through Superdex 200pg, wherein the mobile phase comprises 10mM PB, 200mM Na-citrate, 1% SKL and pH7.4. The sample was left at RT (room temperature) at 4 ℃ for 0,1, 2, 3 and 4 days, and 0.5ml of the sample was injected, and the stability of the sample was measured, and the results are shown in tables 3 and 4. Simultaneously, repeatedly freezing and thawing each group of samples for 1, 2, 3 and 4 times, and measuring the purity of the samples after each freezing and thawing; the repeated freezing and thawing is a common technical means in the field and is not described in detail herein.
TABLE 2 specific parameter conditions for different renaturations
Renaturation buffer type Whether or not desalination is carried out before concentration
Control group 10mM PB，pH7.4 Whether or not
Na-citrate group 10mM PB，200mM Na-citrate，pH7.4 Is that
Group Arg 50mM Tris-HCl，0.5M Arg，10mM GSH，1mM GSSG，pH9.0 Is that
SKL group 10mM PB，200mM Na-citrate，0.5％SKL，pH7.4 Is that
TABLE 3 stability of samples at different temperatures
Figure BDA0002572486520000091
TABLE 4 stability of samples at different freeze-thaw times
Figure BDA0002572486520000092
Figure BDA0002572486520000101
(1) In the experimental process, samples of Arg groups incubated by SKL do not obtain stable monomers after SEC, and the rest three groups, namely a control group, a Na-Citrate group and a SKL group, obtain stable monomers; (2) the stability of the sample is better along with the increase of time at the same temperature, but the stability of the sample is worse when the temperature of the sample is higher; (3) in the freeze-thaw stability experiment, the Na-Citrate group, and the SKL group in the control group have poor effect. It should be noted that the same conclusion can be drawn by performing the above experiments on the APO-J protein formed by the amino acid sequences shown in SEQ ID Nos. 1-9 and 11-28.
3.2 Metal ion Cu2+Effect on renaturation proteins
100 μ l of the renatured protein was taken and the corresponding CuCl was added according to Table 5 below2Placing in a 25 deg.C stabilization box, taking out after 16hr, adding EDTA with final concentration of 0.1mM to stop reaction, adding SDS-PAGE buffer solution into the prepared sample, and performing SDS-PAGE analysis, wherein the result is shown in FIG. 9 (Lane 1: Marker; Lane 2: control; Lane 3: control + CuCl)2(ii) a Lane 4: Na-Citrate group; lane 5: Na-Citrate group + CuCl2(ii) a Lane 6: group L-Arg; lane 7: L-Arg group + CuCl2(ii) a Lane 8: SKL; lane 9: SKL group + CuCl2)。
TABLE 5 addition of CuCl for different groups2Reaction system of
Figure BDA0002572486520000102
As can be seen from FIG. 9, CuCl was added to the renatured APO-J protein2Aggregate formation can be promoted.
Example 4 Another renaturation method of recombinant apolipoprotein J
Taking two expressed proteins (1# and 2#, the corresponding amino acid sequences are SEQ ID. NO3 and 5 respectively), carrying out thallus crushing, inclusion body washing and dissolving, anion chromatography, desalting and liquid changing, cation chromatography, diluting and renaturing according to the following methods respectively, and detecting the stability of the proteins.
The expressed recombinant apolipoprotein #, 2#, was reconstituted with the diluent renaturation solution of example 3 (10mM PB, 200mM Na-citrate, 0.5%SKL, ph7.4) and method for dilution and renaturation for 40h, concentrating (about 10-15 times) the solution for 5h, desalting 6 times by G25 column chromatography to make SKL concentration equal to 0 and make the final concentration of the protein sample about 1.5 mg/mL. The sample was divided into two portions, one of which was added with CuCl2About 3mmol/L, sterile-filtered, incubated at 25 ℃ for 16h, another directly sterile-filtered, incubated at 25 ℃ for 16h, and sampled for non-reducing SDS-PAGE electrophoresis detection, and the stability was examined, the results are shown in tables 6 and 7.
TABLE 6 sample purity under different treatment conditions
Figure BDA0002572486520000111
TABLE 7 results of repeated tests under different treatment conditions
Figure BDA0002572486520000112
As can be seen from tables 6 and 7, CuCl was added2Is beneficial to the stability of the target protein. SKL in the buffer solution can be replaced by detergents such as SDS, the concentration range of the detergents can be 0.15-3.5%, and the renaturation effect is basically the same; the incubation time may range from 20-72 hours, with extension of the incubation time slightly increasing the renaturation rate; desalting by G25 column chromatography can be carried out by ultrafiltration, dialysis, ethanol precipitation, isoelectric point precipitation, salting-out precipitation, etc.
Example 5A recombinant APO-J purification method
65ml of inclusion body containing the amino acid sequence of SEQ ID.NO3 (protein No. 3) was purified at a concentration of 2.78mg/ml (from 03292017 batches of fermentation samples);
1.1 anion exchange chromatography
Column chromatography was performed using POROS HQ, washing with buffer A (50mM Gly-NaOH, 8M Urea, 10mM DDT, pH9.0), and column washing with buffer B (50mM Gly-NaOH, 8M Urea, 10mM DDT, 1M NaCl, pH9.0) and collection.
1.2 desalting and replacing liquid
(1) Connecting the pipeline, the pressure meter and the membrane, rinsing with a buffer solution at a flow rate of 200LMH, exhausting and filling the membrane with the buffer solution; (2) putting the inlet into a sample, controlling the transmembrane pressure to be less than 10psi, concentrating to the concentration of 3mg/ml, and then changing the solution for 5CV until the pH value of the filtrate is consistent with that of the buffer solution; (3) the reflux valve is fully opened, the flow rate is increased, and the air pushes out all the samples.
1.3 cation exchange chromatography
Column chromatography was performed using POROS XS, washing was performed with buffer A (20mM sodium acetate, 8M urea, 10mM DDT, pH5.5), and then with buffer B (20mM sodium acetate, 8M urea, 10mM DDT, 1M NaCl, pH5.5), and collection was performed at a sample concentration of 1.8mg/ml with a yield of 75%.
1.4 renaturation
(1) Adding 0.175ml of 2M DTT into 35ml of the collected liquid of the cation chromatography; (2) 15-fold (V/V) dilution buffer (10mM PB, 200mM Na-citrate, 5mM GSH, 5mM cysteine, pH7.4) was slowly added to the stirred sample to reduce the DTT concentration to 1.25 mM; standing at 4 deg.C for 16hr for renaturation; (3) TFF concentrate the exchange into buffer 1(10mM PB, pH7.4) to give 5 ml.
1.5 SEC purification
Loading the sample after dilution renaturation according to 3-5% of the volume of the SEC column, eluting by using 10mM PB buffer solution, combining the parts with the highest purity of the recombinant apolipoprotein J in the eluted sample, and finally ensuring that the endotoxin and the purity of the target protein meet the requirements; the HCP proportion is 0.000044%, which is less than the 0.1% content standard required by the regulation.
Example 6 toxicological experiments
1. Experimental Material
1.1 Experimental animals mice C57BL/6J (SPF grade), 20 mice, weighing approximately 25g, male. The animal experiment ethics committee of the hospital outside the house approves the use of 20 animals, and the animal experiment ethics batch number: 0077-M-20-HX (X).
1.2 the purification grade of the main reagent recombinant apolipoprotein J (the primary sequence is SEQ ID. NO17, namely No. 17 protein) is PAGE grade, the preparation method is adopted in the embodiment 1, and then the sterile effect is finally achieved by the treatment of 0.22 bacterial filter membrane.
2. Grouping and treatment
The study is developed in the barrier environment of animal centers in Beijing Fuweisan Hospital, a random grouping test is adopted, 20 healthy mice are randomly divided into 5 cages before the experiment, 4 mice are fed in each cage for 6 days in an adaptive manner; and the ears of the mice are punched and numbered, wherein the numbers 001-.
In the 2-4 weeks of the experiment, 2.5% chloral hydrate is selected as an anesthetic, and general anesthesia is carried out by means of intraperitoneal administration, wherein the administration dose is 0.12ml/10g (mouse body weight). The injection is administered by jugular vein injection (when in administration, the mouse is in an anaesthetized state), the administration doses of experimental groups are respectively 10, 40, 160 and 640 mu g of target protein/25 g (the weight of the mouse), the administration is carried out once a week for 3 continuous weeks, the daily behavior and activity of the mouse and the weight, diet, excrement and the like of the mouse are observed and recorded, whether adverse reaction or immunogenic reaction and tolerance occur after the injection of the medicine is emphatically observed, and the specific experimental flow is shown in figure 10.
The possible toxic and adverse reactions of the animals were classified into 4 classes, general reactions (including hair treeing, bradykinesia, disinhibition and loss of appetite), more severe reactions (including cyanosis of the extremities, cyanosis of the nose and puffiness of the extremities), severe reactions (dyspnea) and most severe reactions (death).
3. Results of the experiment
In the experimental process, all mice have normal appearance (hair is bright, movement is rapid, alertness is not different, and feces are normal), and three levels of adverse reactions including 'relatively severe reaction', 'severe reaction' and 'most severe reaction' do not occur; at the end of the experiment, 14 mice were eventually alive, but 6 dead mice were all caused by the surgical procedure; the surviving mice had normal activities of daily behavior and no abnormal appearance.
Furthermore, in the second week of injection, the venous blood vessels were found to be redder and darker in the 40, 160 μ g group than in the placebo group, indicating a higher oxygen content in the venous blood; and the mice in the 10, 40, 160 ug group were more active, more exercise and heavier in weight compared to the placebo group at the third week of injection (i.e., after the third dose), as shown in table 8. After the experiment, the remaining 14 mice were dissected and had clear and normal appearance of the heart, liver, spleen, lung, kidney and other organs.
Table 8 mouse body weight record table
Figure BDA0002572486520000141
Figure BDA0002572486520000151
The size relationship of the average body weight of mice in different groups during the 9.15-9.22 period is 160 μ g group >40 μ g group >10 μ g group > placebo group >640 μ g group; size relationship of average body weight of mice in different groups during 9.26-9.29 160 μ g group >10 μ g group >40 μ g group > placebo group >640 μ g group.
4. Conclusion of the experiment
(1) The appearance and daily behavior of the mice of different drug dose groups (10, 40, 160 and 640 mu g groups) are not different from those of the placebo group, namely the hair is bright, the movement is rapid, the alertness is good, the diet and drinking water are normal, the cyanosis phenomenon does not exist at the mouth and nose, the edema phenomenon of limbs does not exist, the toxic and side effects such as dyspnea do not exist, the mice of different drug dose groups are not obviously uncomfortable, and the apolipoprotein J drug has no toxic and side effects;
(2) after the second jugular vein administration (i.e., day 19/9), mice in the 40, 160 μ g group had venous blood color that was redder and darker than those in the placebo group, indicating higher oxygen content in the venous blood. Meanwhile, the mice in the 10, 40 and 160 mug groups were significantly more active in behavior, healthier and heavier than those in the placebo group during the period from 28 days at 9 months to 30 days at 9 months.
(3) The ultra-high dose (namely 640 mu g group) can cause mild adverse reaction, which is mainly reflected in that the weight of the mice is light, the mice are more sensitive to narcotics, the activity of the mice after the first administration is slightly lower than that of a placebo group, but the appearance of the mice is not obviously different, and the phenomenon of obvious discomfort does not exist; however, this was significantly improved after 2 to 3 days of administration, and the activity of the mice in the 640. mu.g group was not different from that in the placebo group by 6 to 7 days of administration.
It should be noted that similar conclusions can be obtained by performing the toxicity test on recombinant apolipoproteins corresponding to other sequences (SEQ ID Nos. 1-16, 18-28, SEQ ID Nos. 29-36 and SEQ ID Nos. 37-38) in the present invention, and the details are not repeated herein.
Example 7 pharmacodynamic experiment
1. Experimental Material
1.1 Experimental animals Male C57BL ApoJ knockdown atherosclerosis model mice (SPF grade), 20, weighing approximately 25 grams, were purchased ex vivo.
1.2 the purification grade of the main reagent recombinant apolipoprotein J (the primary sequence is SEQ ID. NO21, namely No. 21 protein) is PAGE grade, the preparation method is adopted in the embodiment 1, and then the sterile effect is finally achieved by the treatment of 0.22 bacterial filter membrane.
2. Experimental methods
40 model mice were randomly divided into 4 cages of 10 mice each, into a low dose group (5. mu.g/25 g mice), a medium dose group (20. mu.g/25 g mice), a high dose group (50. mu.g/25 g mice) and a saline control group, adaptively fed for 1 week, then intravenously administered after 2-16 weeks with gas anesthesia, once per week, two animals were taken every 4 weeks for testing, and then dissected for pathological section observation.
3. Results of the experiment
(1) The mice of the medium dose group (with a concentration of the pharmaceutical protein in the blood of 20 μ g/ml) showed an increase in the left ventricular ejection fraction after 4 weeks of administration, increasing in amplitude from 10-12% after 4 weeks to 20-25% after 16 weeks, compared to the saline control group; (2) compared with a normal saline control group, the mice of different dosage groups have obvious reduction of intravascular sediments after 4 weeks, and the reduction range from 4 weeks is 25-30% to 16 weeks is 50-60%; meanwhile, heart color ultrasonography of mice of different dose groups showed significant improvement in cardiac function. That is to say: the medicament to be tested (protein No. 21) has clinical effect of treating chronic heart failure on atherosclerosis model mice. It should be noted that similar conclusions can be obtained by performing the pharmacodynamic experiment on the recombinant apolipoproteins corresponding to other sequences (consisting of SEQ ID NO 1-20, 21-28, SEQ ID NO29-36 and SEQ ID NO 37-38) in the present invention, and the details are not repeated herein.
Example 8 protection of AopB100 by recombinant apolipoprotein J
1. Experimental Material
ApoB 100: loyang Baitai Biotechnology Ltd, concentration 3.3 mg/ml.
Sample 1: dissolving a recombinant apolipoprotein J sample (protein No. 20) with a GSH buffer solution to a concentration of 1.7 mg/ml;
sample 2: the recombinant apolipoprotein J sample (protein No. 20) is dialyzed ((1:500 dialysate, dialyzed for 2 times, each time for 3 hours), and the dialyzed sample is dissolved to the concentration of 1.7mg/ml by using GSH buffer solution;
sample 3: dissolving a recombinant apolipoprotein J sample (protein No. 21) with a GSH buffer solution to the concentration of 2.2 mg/ml;
BSA: 1mg/ml, dissolved in 10mM PB, pH 8.0; PB solution: pH8.0,10 mM.
2. Experimental methods
Detecting the aggregation inhibiting effect on ApoB100 by utilizing the molecular chaperone activity of the recombinant apolipoprotein J, and specifically, heating an ApoB100 solution to 70 ℃ for 5 minutes, and measuring OD360 to check the thermal aggregation degree as a control; ApoB100 was mixed with recombinant apolipoprotein J (sample 1, sample 2, sample 3), heated to 70 ℃ for 5 minutes, and OD360 was measured and loaded as in tables 9 and 10 below.
TABLE 9 sample addition volumes for each group
Figure BDA0002572486520000171
TABLE 10 final post-loading concentrations for each group
Figure BDA0002572486520000172
3. Results of the experiment
TABLE 11 Absorbance (OD360) and aggregation inhibition ratio of samples before and after heating
Figure BDA0002572486520000181
As can be seen from table 11, recombinant apolipoprotein J is effective in inhibiting aggregation of ApoB100 under heating, compared to BSA.
Example 9 protection of CPK protein by recombinant Apolipoprotein J
1. Principle of experiment
Phosphocreatine kinase (CPK protein) is selected as a substrate for detecting the activity of the molecular chaperone, and ApoJ protein is taken as potential molecular chaperone protein. After the CPK protein in the control group is heated to 60 ℃, the original regular peptide chain structure of the CPK protein is opened to be in a loose irregular structure, the hydrophobic group is exposed and then condensed into irregular protein aggregation, the CPK protein is finally denatured, the maximum light absorption of the aggregated protein is realized at 360nm, the light absorption value is positively correlated with the protein denaturation aggregation degree, and therefore the change process can be detected under the wavelength of 360 nm. Apolipoprotein J (apo J), the first protein found to function as a chaperone in vitro, prevents incorrect interactions within or with polypeptide chains in an unnatural state, and aids in correct folding of the protein. Studies show that the ApoJ protein can inhibit the protein denaturation and aggregation of starch metabolism related pathways, including proteins such as glutathione transferase (GST), Creatine Phosphokinase (CPK), Catalase (CAT) and the like, namely, the ApoJ protein can be combined with receptor protein to form an oligomer state, the space stability of the receptor protein is kept, and the misfolding of the receptor protein caused by environmental factors is reduced.
Under the catalysis of creatine kinase, creatine phosphate reacts with ADP to generate creatine and ATP, and the creatine is combined with diacetyl and alpha-naphthol to generate red compounds. The red compound has a linear relation with the creatine content in a certain range, and is compared with creatine standard solution treated by the same method at the wavelength of 520nm to obtain the activity of CK.
2. Experimental Material
Sample 4: dissolving a recombinant apolipoprotein J sample (protein No. 19) with a GSH buffer (namely 20mM PB buffer, 200mM sodium citrate, 5mM GSH, 5mM Cysteine, pH7.4) to the concentration of 2.28 mg/ml;
sample 5: recombinant apolipoprotein J sample (protein No. 20) dissolved to concentration of 1.48mg/ml by GSH buffer solution
Sample 6: a lyophilized powder of apolipoprotein J (i.e., wild-type apolipoprotein J) was dissolved in 10mM PB buffer solution (pH 8.0), and then dialyzed on ice for 2 times (1:500) against 10mM PH8.0 PB buffer solution for 3 hours each to give a final protein concentration of 0.5mg/ml
3. Experimental methods
For samples 4, 5 and 6, experiments were performed in three groups, i.e., group i-only substrate CPK; group II-substrate CPK + BSA protein; group III-substrate CPK + ApoJ protein, the specific concentration is shown in Table 12, CPK enzyme activity before heating is detected;
heating the three groups of samples at 60 ℃ for 5min, then respectively taking out 15ul of samples, and adding the samples into a reaction system for detecting the CPK protease activity; and (3) adjusting the wavelength of the ultraviolet spectrophotometer preheated for 20 minutes to 520nm, detecting the enzyme activity of the heated CPK, and reflecting the protective effect of the target protein ( samples 4, 5 and 6) on the enzyme activity of the CPK according to the change of the enzyme activity. The CPK enzyme activity determination takes Cys and GSH as reflecting substrates, and the detection method is the prior art and is not repeated herein.
TABLE 12 protein, substrate concentrations in different groups
Figure BDA0002572486520000191
Figure BDA0002572486520000201
Remarking: the target proteins are respectively corresponding to the samples 4, 5 and 6
The CPK protease activity reaction system is respectively configured according to different experimental requirements, such as CPK: sample 4 ═ 1.0: the reaction system of 1.0 is shown in Table 13.
TABLE 13 sample addition volume in each reaction system for sample 4
Figure BDA0002572486520000202
4. Results of the experiment
TABLE 14 inhibition of protein aggregation and protection of CPK Activity of samples 4 and 5
Figure BDA0002572486520000203
TABLE 15 inhibition of protein aggregation and protection of CPK Activity of samples 4 and 6
Figure BDA0002572486520000211
Recombinant apolipoprotein J is effective in inhibiting CPK aggregation under heating relative to BSA. Relative to groups I and II, CPK protein in group III with recombinant apolipoprotein J (i.e., sample 4, sample 5) was enzymatically active even after 5 minutes at 60 deg.C; however, the enzyme activity of CPK added to apolipoprotein J (sample 6) after heating decreased to a value below 0, indicating that apolipoprotein J (sample 6) failed to protect the CPK protease activity. That is to say: the sample 4 (protein No. 19) and the sample 5 (protein No. 20) have the activity of inhibiting CPK protein aggregation similar molecular chaperones; while there was no significant difference in the protection of enzyme activity between the sample 4 (protein No. 19) and the sample 5 (protein No. 20), apolipoprotein J (sample 6) was completely devoid of activity for protecting enzyme activity.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Figure BDA0002572486520000221
Figure BDA0002572486520000231
Figure BDA0002572486520000241
Figure BDA0002572486520000251
Figure BDA0002572486520000261
Figure BDA0002572486520000271
Figure BDA0002572486520000281
Figure BDA0002572486520000291
Figure BDA0002572486520000301
Figure BDA0002572486520000311
Figure BDA0002572486520000321
Figure BDA0002572486520000331
Figure BDA0002572486520000341
Figure BDA0002572486520000351
Figure BDA0002572486520000361
Figure BDA0002572486520000371
Figure BDA0002572486520000381
Figure BDA0002572486520000391
Figure BDA0002572486520000401
Figure BDA0002572486520000411
Figure BDA0002572486520000421
Figure BDA0002572486520000431
Figure BDA0002572486520000441
Figure BDA0002572486520000451
Figure BDA0002572486520000461
Figure BDA0002572486520000471
Figure BDA0002572486520000481
Figure BDA0002572486520000491
Figure BDA0002572486520000501
Figure BDA0002572486520000511
Figure BDA0002572486520000521
Figure BDA0002572486520000531
Figure BDA0002572486520000541
Figure BDA0002572486520000551
Figure BDA0002572486520000561
Figure BDA0002572486520000571
Figure BDA0002572486520000581
Figure BDA0002572486520000591
Figure BDA0002572486520000601
Figure BDA0002572486520000611
Figure BDA0002572486520000621
Figure BDA0002572486520000631
Figure BDA0002572486520000641
Figure BDA0002572486520000651
Figure BDA0002572486520000661
Figure BDA0002572486520000671
Figure BDA0002572486520000681
Figure BDA0002572486520000691
Figure BDA0002572486520000701
Figure BDA0002572486520000711
Figure BDA0002572486520000721
Figure BDA0002572486520000731
Figure BDA0002572486520000741
Figure BDA0002572486520000751
Figure BDA0002572486520000761
Figure BDA0002572486520000771
Figure BDA0002572486520000781
Figure BDA0002572486520000791
Figure BDA0002572486520000801
Figure BDA0002572486520000811
Figure BDA0002572486520000821
Figure BDA0002572486520000831
Figure BDA0002572486520000841
Figure BDA0002572486520000851
Figure BDA0002572486520000861
Figure BDA0002572486520000871
Figure BDA0002572486520000881
Figure BDA0002572486520000891
Figure BDA0002572486520000901
Figure BDA0002572486520000911
Figure BDA0002572486520000921
Figure BDA0002572486520000931
Figure BDA0002572486520000941
Figure BDA0002572486520000951
Figure BDA0002572486520000961
Figure BDA0002572486520000971
Figure BDA0002572486520000981
Figure BDA0002572486520000991
Figure BDA0002572486520001001
Figure BDA0002572486520001011
Figure BDA0002572486520001021
Figure BDA0002572486520001031
Figure BDA0002572486520001041
Figure BDA0002572486520001051
Figure BDA0002572486520001061
Figure BDA0002572486520001071
Figure BDA0002572486520001081
Figure BDA0002572486520001091
Figure BDA0002572486520001101
Figure BDA0002572486520001111
Figure BDA0002572486520001121
Figure BDA0002572486520001131
Figure BDA0002572486520001141
Figure BDA0002572486520001151
Figure BDA0002572486520001161
Figure BDA0002572486520001171
Figure BDA0002572486520001181
Figure BDA0002572486520001191
Figure BDA0002572486520001201
Figure BDA0002572486520001211
Figure BDA0002572486520001221
Figure BDA0002572486520001231
Figure BDA0002572486520001241
Figure BDA0002572486520001251
Figure BDA0002572486520001261
Figure BDA0002572486520001271
Figure BDA0002572486520001281
Figure BDA0002572486520001291
Figure BDA0002572486520001301
Figure BDA0002572486520001311
Figure BDA0002572486520001321
Figure BDA0002572486520001331
Figure BDA0002572486520001341
Figure BDA0002572486520001351
Figure BDA0002572486520001361
Figure BDA0002572486520001371
Figure BDA0002572486520001381
Figure BDA0002572486520001391
Figure BDA0002572486520001401
Figure BDA0002572486520001411
Figure BDA0002572486520001421
Figure BDA0002572486520001431
Figure BDA0002572486520001441
Figure BDA0002572486520001451
Figure BDA0002572486520001461
Figure BDA0002572486520001471
Figure BDA0002572486520001481
Figure BDA0002572486520001491
Figure BDA0002572486520001501
Figure BDA0002572486520001511
Figure BDA0002572486520001521
Sequence listing
<110> Xiamen Dengxin Shang product medical science and technology Co., Ltd
<120> crystal structure of recombinant apolipoprotein J or analogue thereof and application
<130> 2018
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
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Met Met Lys Thr Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu
1 5 10 15
Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln
20 25 30
Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn
35 40 45
Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn
50 55 60
Glu Glu Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu Thr
65 70 75 80
Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu
85 90 95
Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr
100 105 110
Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val
115 120 125
Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe
130 135 140
Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln
145 150 155 160
Gln Thr His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser
165 170 175
Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro
180 185 190
Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro
195 200 205
His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe
210 215 220
Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu
225 230 235 240
Glu Met Ile His Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser
245 250 255
Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp
260 265 270
Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu
275 280 285
Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp
290 295 300
Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp
305 310 315 320
Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu
325 330 335
Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu
340 345 350
Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr
355 360 365
Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His
370 375 380
Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys
385 390 395 400
Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser
405 410 415
Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln
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Glu Tyr Arg Lys Lys His Arg Glu Glu
435 440
<210> 2
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<213> Artificial sequence (Artificial sequence)
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Met Met Lys Thr Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu
1 5 10 15
Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln
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Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn
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Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn
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Glu Glu Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu Thr
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Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu
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Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr
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Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val
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Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe
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Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln
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Gln Thr His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser
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Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro
180 185 190
Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro
195 200 205
His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe
210 215 220
Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu
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Glu Met Ile His Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser
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Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp
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Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu
275 280 285
Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp
290 295 300
Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp
305 310 315 320
Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu
325 330 335
Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu
340 345 350
Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr
355 360 365
Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His
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Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys
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Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser
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Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln
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Glu Tyr Glu Glu
435
<210> 3
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Met Met Lys Thr Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu
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Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln
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Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn
35 40 45
Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn
50 55 60
Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys
65 70 75 80
Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys
85 90 95
Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu
100 105 110
Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val
115 120 125
Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu
130 135 140
Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp
145 150 155 160
Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met
165 170 175
Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln
180 185 190
Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro
195 200 205
Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg
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Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe
225 230 235 240
His Ala Met Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln
245 250 255
Ala Met Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr
260 265 270
Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile
275 280 285
Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys
290 295 300
Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln
305 310 315 320
Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg
325 330 335
Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met
340 345 350
Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp
355 360 365
Val Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu
370 375 380
Arg Val Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser
385 390 395 400
Gly Val Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr
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Val Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu
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Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Glu Glu
435 440
<210> 4
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Met Met Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu Ser Gly
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Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met
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Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val
35 40 45
Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu
50 55 60
Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys Lys Glu
65 70 75 80
Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu
85 90 95
Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys
100 105 110
Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg
115 120 125
Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln
130 135 140
Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu
145 150 155 160
Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp
165 170 175
His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg
180 185 190
Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser
195 200 205
Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val
210 215 220
Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala
225 230 235 240
Met Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala Met
245 250 255
Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe
260 265 270
Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His
275 280 285
Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg
290 295 300
Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys
305 310 315 320
Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr
325 330 335
Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn
340 345 350
Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser
355 360 365
Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val
370 375 380
Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val
385 390 395 400
Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr
405 410 415
Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val
420 425 430
Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu Glu
435 440 445
<210> 5
<211> 439
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 5
Met Met Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu Ser Gly
1 5 10 15
Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met
20 25 30
Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val
35 40 45
Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu
50 55 60
Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu Thr Arg Glu
65 70 75 80
Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met
85 90 95
Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met
100 105 110
Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg
115 120 125
Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met
130 135 140
Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr
145 150 155 160
His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile
165 170 175
Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp
180 185 190
Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe
195 200 205
Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser Pro
210 215 220
Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu Met
225 230 235 240
Ile His Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser Pro Ala
245 250 255
Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg
260 265 270
Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met
275 280 285
Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp Cys Ser
290 295 300
Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser
305 310 315 320
Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys
325 330 335
Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu
340 345 350
Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr Gln Gly
355 360 365
Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His Thr Ser
370 375 380
Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys Leu Phe
385 390 395 400
Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser Arg Lys
405 410 415
Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr
420 425 430
Arg Lys Lys His Arg Glu Glu
435
<210> 6
<211> 434
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 6
Met Met Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu Ser Gly
1 5 10 15
Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met
20 25 30
Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val
35 40 45
Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu
50 55 60
Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu Thr Arg Glu
65 70 75 80
Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met
85 90 95
Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met
100 105 110
Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg
115 120 125
Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met
130 135 140
Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr
145 150 155 160
His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile
165 170 175
Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp
180 185 190
Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe
195 200 205
Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser Pro
210 215 220
Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu Met
225 230 235 240
Ile His Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser Pro Ala
245 250 255
Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg
260 265 270
Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met
275 280 285
Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp Cys Ser
290 295 300
Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser
305 310 315 320
Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys
325 330 335
Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu
340 345 350
Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr Gln Gly
355 360 365
Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His Thr Ser
370 375 380
Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys Leu Phe
385 390 395 400
Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser Arg Lys
405 410 415
Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr
420 425 430
Glu Glu
<210> 7
<211> 442
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 7
Met Met Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu Ser Gly
1 5 10 15
Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met
20 25 30
Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val
35 40 45
Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu
50 55 60
Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys Lys Glu
65 70 75 80
Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu
85 90 95
Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys
100 105 110
Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg
115 120 125
Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln
130 135 140
Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu
145 150 155 160
Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp
165 170 175
His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg
180 185 190
Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser
195 200 205
Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val
210 215 220
Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala
225 230 235 240
Met Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala Met
245 250 255
Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe
260 265 270
Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His
275 280 285
Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg
290 295 300
Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys
305 310 315 320
Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr
325 330 335
Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn
340 345 350
Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser
355 360 365
Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val
370 375 380
Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val
385 390 395 400
Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr
405 410 415
Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val
420 425 430
Ala Glu Lys Ala Leu Gln Glu Tyr Glu Glu
435 440
<210> 8
<211> 440
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 8
Met Met Lys Thr Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln Thr
1 5 10 15
Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr
20 25 30
Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys
35 40 45
Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn
50 55 60
Leu Glu Glu Ala Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu Thr Arg
65 70 75 80
Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr
85 90 95
Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys
100 105 110
Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly
115 120 125
Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp
130 135 140
Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln
145 150 155 160
Thr His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser
165 170 175
Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln
180 185 190
Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His
195 200 205
Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser
210 215 220
Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu
225 230 235 240
Met Ile His Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser Pro
245 250 255
Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp
260 265 270
Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg
275 280 285
Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp Cys
290 295 300
Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu
305 310 315 320
Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu
325 330 335
Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln
340 345 350
Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr Gln
355 360 365
Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His Thr
370 375 380
Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys Leu
385 390 395 400
Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser Arg
405 410 415
Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln Glu
420 425 430
Tyr Arg Lys Lys His Arg Glu Glu
435 440
<210> 9
<211> 432
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 9
Met Met Lys Thr Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln Thr
1 5 10 15
Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr
20 25 30
Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys
35 40 45
Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn
50 55 60
Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu
65 70 75 80
Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys
85 90 95
Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys
100 105 110
Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn
115 120 125
Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser
130 135 140
Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln
145 150 155 160
Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp
165 170 175
Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe
180 185 190
Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile
195 200 205
Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His
210 215 220
Ala Met Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala
225 230 235 240
Met Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr Glu
245 250 255
Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg
260 265 270
His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys
275 280 285
Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala
290 295 300
Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu
305 310 315 320
Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu
325 330 335
Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val
340 345 350
Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg
355 360 365
Val Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly
370 375 380
Val Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val
385 390 395 400
Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr
405 410 415
Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu Glu
420 425 430
<210> 10
<211> 427
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 10
Met Met Lys Thr Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln Thr
1 5 10 15
Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr
20 25 30
Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys
35 40 45
Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn
50 55 60
Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu
65 70 75 80
Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys
85 90 95
Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys
100 105 110
Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn
115 120 125
Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser
130 135 140
Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln
145 150 155 160
Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp
165 170 175
Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe
180 185 190
Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile
195 200 205
Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His
210 215 220
Ala Met Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala
225 230 235 240
Met Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr Glu
245 250 255
Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg
260 265 270
His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys
275 280 285
Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala
290 295 300
Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu
305 310 315 320
Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu
325 330 335
Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val
340 345 350
Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg
355 360 365
Val Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly
370 375 380
Val Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val
385 390 395 400
Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr
405 410 415
Val Ala Glu Lys Ala Leu Gln Glu Tyr Glu Glu
420 425
<210> 11
<211> 435
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 11
Met Met Lys Thr Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln Thr
1 5 10 15
Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr
20 25 30
Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys
35 40 45
Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn
50 55 60
Leu Glu Glu Ala Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu Thr Arg
65 70 75 80
Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr
85 90 95
Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys
100 105 110
Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly
115 120 125
Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp
130 135 140
Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln
145 150 155 160
Thr His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser
165 170 175
Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln
180 185 190
Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His
195 200 205
Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser
210 215 220
Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu
225 230 235 240
Met Ile His Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser Pro
245 250 255
Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp
260 265 270
Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg
275 280 285
Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp Cys
290 295 300
Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu
305 310 315 320
Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu
325 330 335
Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln
340 345 350
Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr Gln
355 360 365
Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His Thr
370 375 380
Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys Leu
385 390 395 400
Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser Arg
405 410 415
Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln Glu
420 425 430
Tyr Glu Glu
435
<210> 12
<211> 441
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 12
Met Met Leu Leu Leu Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln
1 5 10 15
Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys
20 25 30
Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile
35 40 45
Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser
50 55 60
Asn Leu Glu Glu Ala Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu Thr
65 70 75 80
Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu
85 90 95
Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr
100 105 110
Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val
115 120 125
Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe
130 135 140
Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln
145 150 155 160
Gln Thr His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser
165 170 175
Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro
180 185 190
Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro
195 200 205
His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe
210 215 220
Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu
225 230 235 240
Glu Met Ile His Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser
245 250 255
Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp
260 265 270
Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu
275 280 285
Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp
290 295 300
Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp
305 310 315 320
Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu
325 330 335
Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu
340 345 350
Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr
355 360 365
Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His
370 375 380
Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys
385 390 395 400
Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser
405 410 415
Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln
420 425 430
Glu Tyr Arg Lys Lys His Arg Glu Glu
435 440
<210> 13
<211> 433
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 13
Met Met Leu Leu Leu Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln
1 5 10 15
Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys
20 25 30
Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile
35 40 45
Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser
50 55 60
Asn Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys
65 70 75 80
Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu
85 90 95
Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val
100 105 110
Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu
115 120 125
Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp
130 135 140
Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met
145 150 155 160
Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln
165 170 175
Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro
180 185 190
Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg
195 200 205
Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe
210 215 220
His Ala Met Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln
225 230 235 240
Ala Met Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr
245 250 255
Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile
260 265 270
Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys
275 280 285
Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln
290 295 300
Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg
305 310 315 320
Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met
325 330 335
Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp
340 345 350
Val Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu
355 360 365
Arg Val Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser
370 375 380
Gly Val Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr
385 390 395 400
Val Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu
405 410 415
Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu
420 425 430
Glu
<210> 14
<211> 428
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 14
Met Met Leu Leu Leu Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln
1 5 10 15
Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys
20 25 30
Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile
35 40 45
Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser
50 55 60
Asn Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys
65 70 75 80
Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu
85 90 95
Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val
100 105 110
Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu
115 120 125
Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp
130 135 140
Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met
145 150 155 160
Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln
165 170 175
Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro
180 185 190
Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg
195 200 205
Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe
210 215 220
His Ala Met Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln
225 230 235 240
Ala Met Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr
245 250 255
Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile
260 265 270
Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys
275 280 285
Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln
290 295 300
Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg
305 310 315 320
Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met
325 330 335
Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp
340 345 350
Val Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu
355 360 365
Arg Val Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser
370 375 380
Gly Val Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr
385 390 395 400
Val Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu
405 410 415
Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Glu Glu
420 425
<210> 15
<211> 436
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 15
Met Met Leu Leu Leu Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln
1 5 10 15
Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys
20 25 30
Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile
35 40 45
Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser
50 55 60
Asn Leu Glu Glu Ala Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu Thr
65 70 75 80
Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu
85 90 95
Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr
100 105 110
Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val
115 120 125
Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe
130 135 140
Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln
145 150 155 160
Gln Thr His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser
165 170 175
Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro
180 185 190
Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro
195 200 205
His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe
210 215 220
Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu
225 230 235 240
Glu Met Ile His Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser
245 250 255
Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp
260 265 270
Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu
275 280 285
Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp
290 295 300
Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp
305 310 315 320
Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu
325 330 335
Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu
340 345 350
Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr
355 360 365
Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His
370 375 380
Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys
385 390 395 400
Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser
405 410 415
Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln
420 425 430
Glu Tyr Glu Glu
435
<210> 16
<211> 438
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 16
Met Met Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser
1 5 10 15
Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn
20 25 30
Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu
35 40 45
Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu
50 55 60
Glu Ala Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser
65 70 75 80
Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met
85 90 95
Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys
100 105 110
Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln
115 120 125
Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn
130 135 140
Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His
145 150 155 160
Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile
165 170 175
Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr
180 185 190
Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe
195 200 205
Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr
210 215 220
Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu Met Ile
225 230 235 240
His Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser Pro Ala Phe
245 250 255
Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr
260 265 270
Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys
275 280 285
Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr
290 295 300
Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu
305 310 315 320
Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser
325 330 335
Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn
340 345 350
Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu
355 360 365
Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His Thr Ser Asp
370 375 380
Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys Leu Phe Asp
385 390 395 400
Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser Arg Lys Asn
405 410 415
Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg
420 425 430
Lys Lys His Arg Glu Glu
435
<210> 17
<211> 425
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 17
Met Met Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser
1 5 10 15
Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn
20 25 30
Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu
35 40 45
Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Glu Asp
50 55 60
Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro
65 70 75 80
Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro
85 90 95
Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser
100 105 110
Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser
115 120 125
Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu
130 135 140
Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp His
145 150 155 160
Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe
165 170 175
Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu
180 185 190
Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg
195 200 205
Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met
210 215 220
Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala Met Asp
225 230 235 240
Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile
245 250 255
Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn
260 265 270
Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu
275 280 285
Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu
290 295 300
Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg
305 310 315 320
Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr
325 330 335
Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg
340 345 350
Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr
355 360 365
Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr
370 375 380
Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val
385 390 395 400
Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala
405 410 415
Glu Lys Ala Leu Gln Glu Tyr Glu Glu
420 425
<210> 18
<211> 433
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 18
Met Met Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser
1 5 10 15
Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn
20 25 30
Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu
35 40 45
Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu
50 55 60
Glu Ala Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser
65 70 75 80
Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met
85 90 95
Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys
100 105 110
Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln
115 120 125
Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn
130 135 140
Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His
145 150 155 160
Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile
165 170 175
Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr
180 185 190
Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe
195 200 205
Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr
210 215 220
Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu Met Ile
225 230 235 240
His Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser Pro Ala Phe
245 250 255
Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr
260 265 270
Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys
275 280 285
Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr
290 295 300
Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu
305 310 315 320
Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser
325 330 335
Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn
340 345 350
Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu
355 360 365
Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His Thr Ser Asp
370 375 380
Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys Leu Phe Asp
385 390 395 400
Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser Arg Lys Asn
405 410 415
Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Glu
420 425 430
Glu
<210> 19
<211> 429
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 19
Met Met Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn
1 5 10 15
Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly
20 25 30
Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys
35 40 45
Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys Lys Glu Asp Ala
50 55 60
Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly
65 70 75 80
Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys
85 90 95
Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly
100 105 110
Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser
115 120 125
Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu
130 135 140
Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp His Phe
145 150 155 160
Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe
165 170 175
Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro
180 185 190
His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser
195 200 205
Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe
210 215 220
Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala Met Asp Ile
225 230 235 240
His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg
245 250 255
Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser
260 265 270
Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile
275 280 285
Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg
290 295 300
Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys
305 310 315 320
Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser
325 330 335
Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu
340 345 350
Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr
355 360 365
Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu
370 375 380
Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro
385 390 395 400
Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu
405 410 415
Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu Glu
420 425
<210> 20
<211> 421
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 20
Met Met Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn
1 5 10 15
Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly
20 25 30
Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys
35 40 45
Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu
50 55 60
Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala
65 70 75 80
Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe
85 90 95
Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu
100 105 110
Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly
115 120 125
Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met
130 135 140
Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp
145 150 155 160
Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr
165 170 175
His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe
180 185 190
Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu
195 200 205
Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu Met Ile His
210 215 220
Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser Pro Ala Phe Gln
225 230 235 240
His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val
245 250 255
Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp
260 265 270
Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn
275 280 285
Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln
290 295 300
Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr
305 310 315 320
Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu
325 330 335
Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp
340 345 350
Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His Thr Ser Asp Ser
355 360 365
Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys Leu Phe Asp Ser
370 375 380
Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro
385 390 395 400
Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys
405 410 415
Lys His Arg Glu Glu
420
<210> 21
<211> 416
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 21
Met Met Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn
1 5 10 15
Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly
20 25 30
Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys
35 40 45
Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu
50 55 60
Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala
65 70 75 80
Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe
85 90 95
Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu
100 105 110
Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly
115 120 125
Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met
130 135 140
Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp
145 150 155 160
Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr
165 170 175
His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe
180 185 190
Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu
195 200 205
Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu Met Ile His
210 215 220
Glu Ala Gln Gln Ala Met Asp Ile His Phe His Ser Pro Ala Phe Gln
225 230 235 240
His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val
245 250 255
Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp
260 265 270
Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn
275 280 285
Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln
290 295 300
Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr
305 310 315 320
Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu
325 330 335
Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp
340 345 350
Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His Thr Ser Asp Ser
355 360 365
Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys Leu Phe Asp Ser
370 375 380
Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro
385 390 395 400
Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Glu Glu
405 410 415
<210> 22
<211> 424
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 22
Met Met Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn
1 5 10 15
Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly
20 25 30
Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys
35 40 45
Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys Lys Glu Asp Ala
50 55 60
Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly
65 70 75 80
Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys
85 90 95
Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly
100 105 110
Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser
115 120 125
Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu
130 135 140
Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp His Phe
145 150 155 160
Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe
165 170 175
Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro
180 185 190
His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser
195 200 205
Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe
210 215 220
Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala Met Asp Ile
225 230 235 240
His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg
245 250 255
Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser
260 265 270
Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile
275 280 285
Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg
290 295 300
Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys
305 310 315 320
Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser
325 330 335
Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu
340 345 350
Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr
355 360 365
Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu
370 375 380
Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro
385 390 395 400
Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu
405 410 415
Lys Ala Leu Gln Glu Tyr Glu Glu
420
<210> 23
<211> 434
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 23
Met Met Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu
1 5 10 15
Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln
20 25 30
Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr
35 40 45
Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys
50 55 60
Lys Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu
65 70 75 80
Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu
85 90 95
Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg
100 105 110
Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe
115 120 125
Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile
130 135 140
Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val
145 150 155 160
Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe
165 170 175
Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu
180 185 190
Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser
195 200 205
Arg Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn
210 215 220
Phe His Ala Met Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln
225 230 235 240
Gln Ala Met Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro
245 250 255
Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu
260 265 270
Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp
275 280 285
Lys Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser
290 295 300
Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu
305 310 315 320
Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys
325 330 335
Met Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn
340 345 350
Trp Val Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr
355 360 365
Leu Arg Val Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro
370 375 380
Ser Gly Val Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile
385 390 395 400
Thr Val Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met
405 410 415
Glu Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg
420 425 430
Glu Glu
<210> 24
<211> 426
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 24
Met Met Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu
1 5 10 15
Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln
20 25 30
Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr
35 40 45
Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu
50 55 60
Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn
65 70 75 80
Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln
85 90 95
Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu
100 105 110
Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr
115 120 125
Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg
130 135 140
Gln Gln Thr His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala
145 150 155 160
Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu
165 170 175
Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg
180 185 190
Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro
195 200 205
Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe
210 215 220
Leu Glu Met Ile His Glu Ala Gln Gln Ala Met Asp Ile His Phe His
225 230 235 240
Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp
245 250 255
Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys
260 265 270
Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val
275 280 285
Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu
290 295 300
Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu
305 310 315 320
Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu
325 330 335
Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu
340 345 350
Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser
355 360 365
His Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val
370 375 380
Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val
385 390 395 400
Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu
405 410 415
Gln Glu Tyr Arg Lys Lys His Arg Glu Glu
420 425
<210> 25
<211> 421
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 25
Met Met Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu
1 5 10 15
Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln
20 25 30
Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr
35 40 45
Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu
50 55 60
Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn
65 70 75 80
Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln
85 90 95
Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu
100 105 110
Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr
115 120 125
Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg
130 135 140
Gln Gln Thr His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala
145 150 155 160
Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu
165 170 175
Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg
180 185 190
Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro
195 200 205
Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe
210 215 220
Leu Glu Met Ile His Glu Ala Gln Gln Ala Met Asp Ile His Phe His
225 230 235 240
Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp
245 250 255
Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys
260 265 270
Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val
275 280 285
Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu
290 295 300
Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu
305 310 315 320
Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu
325 330 335
Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu
340 345 350
Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser
355 360 365
His Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val
370 375 380
Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val
385 390 395 400
Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu
405 410 415
Gln Glu Tyr Glu Glu
420
<210> 26
<211> 429
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 26
Met Met Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu
1 5 10 15
Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln
20 25 30
Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr
35 40 45
Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys
50 55 60
Lys Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu
65 70 75 80
Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu
85 90 95
Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg
100 105 110
Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe
115 120 125
Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile
130 135 140
Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val
145 150 155 160
Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe
165 170 175
Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu
180 185 190
Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser
195 200 205
Arg Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn
210 215 220
Phe His Ala Met Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln
225 230 235 240
Gln Ala Met Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro
245 250 255
Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu
260 265 270
Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp
275 280 285
Lys Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser
290 295 300
Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu
305 310 315 320
Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys
325 330 335
Met Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn
340 345 350
Trp Val Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr
355 360 365
Leu Arg Val Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro
370 375 380
Ser Gly Val Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile
385 390 395 400
Thr Val Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met
405 410 415
Glu Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Glu Glu
420 425
<210> 27
<211> 449
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 27
Met Met Lys Thr Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu
1 5 10 15
Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln
20 25 30
Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn
35 40 45
Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn
50 55 60
Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys
65 70 75 80
Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys
85 90 95
Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu
100 105 110
Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val
115 120 125
Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu
130 135 140
Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp
145 150 155 160
Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met
165 170 175
Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln
180 185 190
Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro
195 200 205
Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg
210 215 220
Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe
225 230 235 240
His Ala Met Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln
245 250 255
Ala Met Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr
260 265 270
Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile
275 280 285
Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys
290 295 300
Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln
305 310 315 320
Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg
325 330 335
Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met
340 345 350
Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp
355 360 365
Val Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu
370 375 380
Arg Val Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser
385 390 395 400
Gly Val Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr
405 410 415
Val Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu
420 425 430
Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu
435 440 445
Glu
<210> 28
<211> 427
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 28
Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly
1 5 10 15
Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys
20 25 30
Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu
35 40 45
Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys Lys Glu Asp Ala Leu Asn
50 55 60
Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys
65 70 75 80
Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys
85 90 95
Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly
100 105 110
Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe
115 120 125
Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp
130 135 140
Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp His Phe Ser Arg
145 150 155 160
Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg
165 170 175
Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg
180 185 190
Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met
195 200 205
Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro
210 215 220
Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala Met Asp Ile His Phe
225 230 235 240
His Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu Gly
245 250 255
Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly
260 265 270
Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser
275 280 285
Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu
290 295 300
Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn
305 310 315 320
Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu
325 330 335
Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn
340 345 350
Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val Ala
355 360 365
Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val Val
370 375 380
Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu
385 390 395 400
Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala
405 410 415
Leu Gln Glu Tyr Arg Lys Lys His Arg Glu Glu
420 425
<210> 29
<211> 227
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 29
Met Met Lys Thr Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu
1 5 10 15
Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln
20 25 30
Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn
35 40 45
Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn
50 55 60
Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys
65 70 75 80
Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys
85 90 95
Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu
100 105 110
Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val
115 120 125
Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu
130 135 140
Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp
145 150 155 160
Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met
165 170 175
Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln
180 185 190
Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro
195 200 205
Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg
210 215 220
Ile Val Arg
225
<210> 30
<211> 219
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 30
Met Met Lys Thr Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu
1 5 10 15
Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln
20 25 30
Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn
35 40 45
Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn
50 55 60
Glu Glu Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu Thr
65 70 75 80
Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu
85 90 95
Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr
100 105 110
Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val
115 120 125
Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe
130 135 140
Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln
145 150 155 160
Gln Thr His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser
165 170 175
Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro
180 185 190
Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro
195 200 205
His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg
210 215
<210> 31
<211> 225
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 31
Met Met Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu Ser Gly
1 5 10 15
Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met
20 25 30
Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val
35 40 45
Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu
50 55 60
Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys Lys Glu
65 70 75 80
Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu
85 90 95
Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys
100 105 110
Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg
115 120 125
Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln
130 135 140
Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu
145 150 155 160
Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp
165 170 175
His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg
180 185 190
Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser
195 200 205
Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val
210 215 220
Arg
225
<210> 32
<211> 217
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 32
Met Met Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu Ser Gly
1 5 10 15
Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met
20 25 30
Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val
35 40 45
Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu
50 55 60
Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu Thr Arg Glu
65 70 75 80
Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met
85 90 95
Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met
100 105 110
Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg
115 120 125
Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met
130 135 140
Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr
145 150 155 160
His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile
165 170 175
Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp
180 185 190
Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe
195 200 205
Phe Phe Pro Lys Ser Arg Ile Val Arg
210 215
<210> 33
<211> 216
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 33
Met Met Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser
1 5 10 15
Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn
20 25 30
Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu
35 40 45
Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu
50 55 60
Glu Ala Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser
65 70 75 80
Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met
85 90 95
Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys
100 105 110
Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln
115 120 125
Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn
130 135 140
Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His
145 150 155 160
Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile
165 170 175
Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr
180 185 190
Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe
195 200 205
Phe Pro Lys Ser Arg Ile Val Arg
210 215
<210> 34
<211> 208
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 34
Met Met Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser
1 5 10 15
Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn
20 25 30
Lys Glu Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu
35 40 45
Ile Glu Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Glu Asp
50 55 60
Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro
65 70 75 80
Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro
85 90 95
Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser
100 105 110
Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser
115 120 125
Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu
130 135 140
Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp His
145 150 155 160
Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe
165 170 175
Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu
180 185 190
Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg
195 200 205
<210> 35
<211> 207
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 35
Met Met Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn
1 5 10 15
Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly
20 25 30
Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys
35 40 45
Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys Lys Glu Asp Ala
50 55 60
Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly
65 70 75 80
Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys
85 90 95
Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly
100 105 110
Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser
115 120 125
Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu
130 135 140
Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp His Phe
145 150 155 160
Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe
165 170 175
Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro
180 185 190
His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg
195 200 205
<210> 36
<211> 199
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 36
Met Met Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn
1 5 10 15
Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly
20 25 30
Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys
35 40 45
Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu
50 55 60
Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala
65 70 75 80
Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe
85 90 95
Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu
100 105 110
Glu Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly
115 120 125
Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met
130 135 140
Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp
145 150 155 160
Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr
165 170 175
His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe
180 185 190
Pro Lys Ser Arg Ile Val Arg
195
<210> 37
<211> 222
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 37
Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met
1 5 10 15
Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala Met Asp
20 25 30
Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile
35 40 45
Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn
50 55 60
Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu
65 70 75 80
Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu
85 90 95
Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg
100 105 110
Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr
115 120 125
Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg
130 135 140
Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr
145 150 155 160
Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr
165 170 175
Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val
180 185 190
Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala
195 200 205
Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu Glu
210 215 220
<210> 38
<211> 217
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 38
Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met
1 5 10 15
Phe Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala Met Asp
20 25 30
Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile
35 40 45
Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn
50 55 60
Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu
65 70 75 80
Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu
85 90 95
Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg
100 105 110
Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr
115 120 125
Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg
130 135 140
Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr
145 150 155 160
Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr
165 170 175
Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val
180 185 190
Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala
195 200 205
Glu Lys Ala Leu Gln Glu Tyr Glu Glu
210 215

Claims (11)

1. A recombinant apolipoprotein J is characterized in that the primary amino acid sequence of the recombinant apolipoprotein J is any one of SEQ ID NO. 1-28.
2. The recombinant apolipoprotein J according to claim 1, consisting of an A fragment and a B fragment, wherein the amino acid primary sequence of the A fragment is any one of SEQ ID No.29-36, and the amino acid primary sequence of the B fragment is any one of SEQ ID No. 37-38.
3. The recombinant apolipoprotein J according to claim 2, wherein the a and B segments are linked by one or more of hydrogen bonding, disulfide bonding, peptide bonding.
4. The recombinant apolipoprotein J according to claim 1 or 2, wherein the secondary and tertiary structure of the recombinant apolipoprotein J comprises 2-4 domains, 2-5 β folds and 2-5 α helices.
5. The recombinant apolipoprotein J according to claim 4, wherein the molecular structure of the recombinant apolipoprotein J crystal comprises 3 domains, 3 β folds and 2 α helices.
6. A method for preparing a recombinant apolipoprotein J according to any one of claims 1 to 5, comprising cell disruption, inclusion body washing and solubilization, anion chromatography, desalting and liquid exchange, cation chromatography, renaturation and SEC chromatography.
7. The method of claim 6, wherein the recombinant apolipoprotein J is selected from the group consisting of: the renaturation adopts stage dilution, and a detergent with a certain concentration is added while a small molecular chaperone and a redox couple are introduced into a renaturation buffer solution.
8. Use of a recombinant apolipoprotein J according to any one of claims 1-5 for the preparation of a medicament for the prevention and/or treatment of a disease.
9. The use of claim 8, wherein the disease comprises cardiovascular disease, amyloidosis, dry eye.
10. The use according to claim 8, wherein the disease is a gene-deficient disease.
11. Use of the recombinant apolipoprotein J according to any one of claims 1-5 in the preparation of a medicament for treating skin diseases and wounds, a longevity drug, a health drug and an immunity-improving drug.
CN202010644415.8A 2019-07-09 2020-07-07 Crystal structure and application of recombinant apolipoprotein J and analogue thereof Pending CN112210003A (en)

Applications Claiming Priority (4)

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