阅读说明：本技术 肺炎链球菌蛋白PepN在抗过敏性哮喘中的应用 (Application of streptococcus pneumoniae protein PepN in resisting allergic asthma ) 是由 胥文春 吴光英 尹一兵 张雪梅 王虹 何於娟 于 2019-09-18 设计创作，主要内容包括：本发明提供了一种抗过敏性哮喘的肺炎链球菌蛋白PepN及其应用,属于微生物技术领域以及医药技术领域。本发明要解决的技术问题是为过敏性哮喘的防治提供一种新的选择。本发明的技术方案是肺炎链球菌蛋白PepN,其序列如SEQ ID NO.1所示。肺炎链球菌蛋白PepN具有抗过敏性哮喘的作用,具体体现在：(1)显著降低过敏性哮喘小鼠支气管肺泡灌洗液中嗜酸性粒细胞的产生；(2)显著减轻过敏性哮喘小鼠肺部炎症反应；(3)显著降低过敏性哮喘小鼠血清中IgE的产生；(4)显著降低过敏性哮喘小鼠支气管肺泡灌洗液中IL-5和IL-13的水平。因此,肺炎链球菌蛋白PepN在制备抗过敏性哮喘的药品中,具有良好的应用前景。(the invention provides a streptococcus pneumoniae protein PepN for resisting allergic asthma and application thereof, belonging to the technical field of microorganisms and medicines. The invention aims to provide a new choice for preventing and treating allergic asthma. The technical scheme of the invention is that the streptococcus pneumoniae protein PepN has a sequence shown in SEQ ID NO. 1. The streptococcus pneumoniae protein PepN has the function of resisting allergic asthma, and is specifically represented by the following components: (1) the generation of eosinophils in bronchoalveolar lavage fluid of allergic asthma mice is remarkably reduced; (2) the lung inflammatory reaction of the allergic asthma mouse is obviously relieved; (3) the production of IgE in the serum of an allergic asthma mouse is obviously reduced; (4) the level of IL-5 and IL-13 in bronchoalveolar lavage fluid of allergic asthma mice is remarkably reduced. Therefore, the streptococcus pneumoniae protein PepN has good application prospect in preparing anti-allergic asthma medicines.)

1. A streptococcus pneumoniae protein PepN or polypeptide characterized by:

(1) An amino acid sequence shown as SEQ ID NO. 1;

(2) Any of the sequences contains 50 continuous amino acid sequences in (1).

2. Use of the protein or polypeptide of claim 1 for the preparation of a medicament against allergic asthma.

Technical Field

The invention relates to an anti-allergic asthma streptococcus pneumoniae protein and application thereof, belonging to the technical field of microorganisms and medicines.

background

Allergic asthma is a chronic airway inflammatory disease mainly characterized by eosinophil infiltration, excessive Th2 type immune reaction, and increase of allergen-specific IgE due to allergen, and is usually manifested by symptoms of recurrent cough, chest distress, wheezing, etc., which often occur in infants and young children and are accompanied by life-long.

In recent years, about 1 million people suffer from allergic asthma worldwide, and the prevalence rate of allergic asthma is increasing in developed countries; about 1000 million patients with allergic asthma in China exist, and the prevalence rate of the allergic asthma is about 1.3%. Allergic asthma has become one of the most common chronic diseases in the world, and causes great troubles to the health and the economy of the public.

The existing treatment methods of asthma mainly comprise allergen avoidance, drug therapy and allergen-specific immunotherapy, but have the defects of only relieving asthma, limited curative effect, long time and the like, so that the development of a novel therapy for preventing or treating asthma is urgently needed. Epidemiological studies have shown that living in an environment with increased microbial exposure in the early years reduces the risk of developing later allergies and asthma. More and more studies have shown that early bacterial infections, such as helicobacter pylori, lactobacillus rhamnosus, bifidobacteria, etc., inhibit the development of asthma. However, clinical studies on prevention of allergic diseases using lactobacillus rhamnosus and bifidobacterium animalis, which have been found to alleviate symptoms of allergic asthma, have been poor or the effect is not clear. The bacterial components have the advantages of high bioavailability, easy evaluation of dose-response relationship, low clinical response variability, clear physicochemical properties and the like. Therefore, the research and development of the bacterial component capable of inhibiting the allergic asthma have good application prospect in the prevention of the allergic asthma, and a new method is provided for the prevention of the allergic asthma.

Studies have shown that the development of asthma is associated with streptococcus pneumoniae infection. Asthma-related hospitalization rates decreased after immunization of streptococcus pneumoniae vaccines in children, and both live and inactivated streptococcus pneumoniae were shown to inhibit asthma in experimental studies in a mouse model. Thus, streptococcus pneumoniae has certain specific components that inhibit the development of asthma. Streptococcus pneumoniae aminopeptidase N (PepN) is an exoprotease that selectively cleaves amino acid residues from the N-terminus of proteins and polypeptides to yield free amino acids, and has been reported to be an immunosuppressive streptococcus pneumoniae component independent of its peptidase activity. The invention firstly verifies the function of PepN in resisting allergic asthma.

disclosure of Invention

The invention aims to provide a streptococcus pneumoniae protein and application thereof in resisting allergic asthma, and provides a new method for preventing the allergic asthma.

The technical scheme provided by the invention is that the streptococcus pneumoniae protein PepN or polypeptide is characterized in that: (1) an amino acid sequence shown as SEQ ID NO. 1; (2) any of the sequences contains 50 continuous amino acid sequences in (1).

The invention provides a preparation method of streptococcus pneumoniae protein PepN.

The invention provides application of streptococcus pneumoniae protein PepN in resisting allergic asthma.

the invention has the beneficial effects that: the streptococcus pneumoniae protein PepN is verified to have the effect of resisting allergic asthma for the first time, and the method is specifically shown as follows: significantly reduced eosinophil production in bronchoalveor lavagefluid (BALF) in mice with allergic asthma; the lung inflammatory reaction of the allergic asthma mouse is obviously relieved; the production of IgE in the serum of an allergic asthma mouse is obviously reduced; significantly reduces the level of IL-5 and IL-13 in BALF of the allergic asthma mice. Therefore, the streptococcus pneumoniae protein PepN has good application prospect in preventing allergic asthma as a bacterial component, and the invention provides a new choice for preventing allergic asthma.

Drawings

FIG. 1 shows the PCR identification of recombinant plasmid pET28a (+) -PepN and the expression and purification of the protein. Wherein, the A picture is the result of identifying pET28a (+) -PepN: m is DNAmarker, and the band 1 is pET28a (+) -PepN nucleotide sequence identification (2544 bp). Panel B shows the purified expression of PepN: m is protein marker, and band 1 is recombinant PepN (molecular weight 95 KDa).

FIG. 2 is a graph showing the effect of PepN on the total and differential counts of inflammatory cells in BALF of allergic asthma mice.

FIG. 3 is HE staining and PAS staining to observe the effect of PepN on lung tissue inflammatory cell infiltration and mucus production in allergic asthma mice.

FIG. 4 is a graph showing the effect of PepN on total IgE in serum from mice with allergic asthma.

FIG. 5 is a graph showing the effect of PepN on IL-5 and IL-13 levels in BALF of allergic asthma mice.

Detailed Description

The invention is further illustrated with reference to specific examples.

Preparation of Streptococcus pneumoniae aminopeptidase N (PepN)

1 materials of the experiment

Plasmid pET28a (+) was obtained from Novagen, PrimeStar Hi-Fi enzyme for PCR, dNTPs, Buffer, MgCl2 was obtained from Takara Bio Inc. (Dalian) and PTC-200 PCR was a Perkin Elmer product.

2 method of experiment

2.1 construction of the recombinant expression plasmid pET28a (+) -PepN.

2.1.1 design and Synthesis of primers:

The primers were designed using premier5.0 with reference to the entire sequence of genomic DNA of Streptococcus pneumoniae D39 (GeneBank accession No. CP000410.2) as a template, and synthesized by Biotechnology engineering (Shanghai) GmbH.

PepN upstream primer: 5'-GGAATTCCATATGATGCAAGCAGTTGAACATT-3', containing an NdeI site; PepN downstream primer: 5'-CCCTCGAGTTATGCATTTCCGTATTGA-3', containing an XhoI site.

2.1.2 PCR amplification of the Gene of interest:

an amplification system:

Wherein "P1 (5pM) 2. mu.l" means: the primer F-PepN was added in an amount of 2. mu.l at a concentration of 5 pM. "P2 (5pM) 2. mu.l" means: the primer R-PepN was added in an amount of 2. mu.l at a concentration of 5 pM.

Amplification conditions: 10min at 95 ℃, 30s at 55 ℃, 3min at 72 ℃ for 30s and 35 cycles; 10min at 72 ℃ for 1 time. The corresponding target gene is obtained by amplification under the above conditions.

2.1.3 construction of prokaryotic expression vectors

The recovery of PCR products was carried out according to the kit instructions provided by Roche, Inc. (Roche Inc.), the plasmid pET28a (+) was carried out according to the instructions of the Omega miniplasmid DNA extraction kit, then the vector DNA and the foreign DNA were subjected to double digestion and recovery, and finally products were recovered by ligation, and the ligation reaction system was as shown in the following Table 2:

Connection conditions are as follows: after the sample addition was completed, the mixture was mixed well, centrifuged instantaneously, and then ligated in a PCR instrument at 22 ℃ for 20 min.

Transformation of the ligation products into e.coli dh5 α competent cells:

Streaking E.coli DH5 alpha bacteria to inoculate LB plate, and incubating at 37 ℃ overnight (12-14 h);

Picking a single colony and inoculating the single colony in 3ml LB;

adding 100 μ l of LB at 37 deg.C overnight (12-14h) at 200rpm, and adding 2ml of LB at 37 deg.C and 3h at 300 rpm;

Adding 1.5ml of bacterial liquid into an ice-precooled EP tube, carrying out ice bath for 10min, and then collecting thalli at 4000rpm for 5 min;

adding 2150 μ l of precooled 0.1mM CaCl, and collecting thallus at 9000rpm for 2min after heavy suspension;

Adding 2150 μ l of precooled 0.1mM CaCl, and suspending;

Adding 10 μ l of the ligation reaction product, mixing uniformly, and performing ice water bath for 30 min;

thermally shocking at 42 deg.C for 30s, and ice-water bathing for 2 min;

Adding 800 mu lLB culture medium, and resuscitating at 37 ℃ and 100rpm1 h;

Coating 200 mul of bacterial liquid on an LK plate;

Incubate at 37 ℃ for 13 h.

2.1.4 screening and identification of the (+) -PepN recombinant pET28a

8 kanamycin-resistant colonies are picked, respectively placed in 2ml LK (LB containing 50ug/ml Kana) culture media, enriched at 180rpm3h, and subjected to bacteria liquid PCR identification; selecting 1 suspicious positive colony for enrichment, and sending to Beijing Optimalaceae New Biotechnology Limited for bidirectional sequencing.

2.2 prokaryotic expression plasmid pET28a (+) -PepN in Escherichia coli expression, identification and purification.

2.2.1 transformation of the recombinant plasmid pET28a (+) -PepN into the host bacterium BL21(DE 3).

2.2.2 IPTG induces large scale expression of PepN.

2.2.3 purification of recombinant proteins: after ultrasonic bacteria breaking, taking a supernatant of a bacteria breaking solution for purification; at 4 deg.C, 12000rpm for 30min, the supernatant was filtered through a 0.45 μm filter membrane, and the filtrate was collected for further use.

affinity chromatography purification, namely sucking 2ml of 50% Ni2 ⁺ -NTA resin suspension into a chromatographic column, balancing by 20ml of ultrasonic crushing buffer solution, sucking the balanced Ni2 ⁺ -NTA resin suspension out, fully and uniformly mixing with the filtrate, carrying out ice bath for 1h, slightly and uniformly mixing once every 5min, transferring the suspension into the chromatographic column, allowing the liquid to naturally flow out, balancing a column bed, carrying out gradient elution at different imidazole concentrations, and respectively collecting eluates.

2.2.4 Ultrafiltration in PBS to remove imidazole.

2.2.5 quantification of recombinant proteins (Bradford assay)

(1) Sucking 6 μ l of a standard bovine serum albumin solution of 20mg/ml, diluting the solution 40 times to 0.5mg/ml with 0.15mmo1/LNaCl, respectively adding 10 μ l, 20 μ l, 30 μ l and 40 μ l of BSA solution into two groups of 4 test tubes, respectively, then fixing the total volume to 200 μ l with 0.15mmol/LNaCl, and simultaneously taking one test tube and adding only 200 μ l of 0.15mmol/LNaCl for zero adjustment;

(2) Taking 20 μ l of the purified product, and complementing the purified product to a total volume of 200 μ l by 0.15 mmol/LNaCl;

(3) Adding 2ml of Coomassie brilliant blue G-250 dye solution into each tube, shaking, mixing uniformly, and standing at room temperature for 30 min;

(4) Reading A590 value in DU-Series600 full wavelength spectrophotometer, automatically drawing standard curve by instrument and calculating protein content of sample. The purified dialyzed PepN protein concentration was 2mg/ml as determined by Bradford method.

2.2.6 removal and detection of endotoxin in recombinant proteins

Refer to "Chinese pharmacopoeia" (published by Chinese pharmaceutical science and technology Press on 6/2015 and 5/2015), and end-point chromogenic method for detecting bacterial endotoxin. The endotoxin content of each protein is less than 0.1 EU/mu g.

3. Results and analysis of the experiments

3.1 construction of recombinant expression plasmid pET28a (+) -PepN

3.1 construction of recombinant expression plasmid pET28a (+) -PepN

FIG. 1A shows the PCR identification result of recombinant expression plasmid pET28a (+) -PepN, wherein, the band 1 is DNA marker, the band 2 is single PCR band, and is consistent with the target DNA fragment of 2544 bp; in addition, the sequencing results of the PCR products completely matched the expected sequence alignment. The above results confirmed that the target gene fragment was correctly inserted into the expression vector.

3.2 expression, identification and purification of PepN in E.coli

FIG. 1B shows the SDS-PAGE analysis of the recombinant PepN, the band 1 is protein marker, the band 2 is recombinant PepN, the size of the recombinant PepN is consistent with the target protein of about 95KD, and the protein purity is above 90%. The above results confirmed that the target protein was successfully expressed and purified.

Second, PepN has preventive effect on allergic asthma of mice

1 materials of the experiment

The experimental animals are 6-8 week old female BABL/c mice purchased from animal experiment center of Chongqing medical university. The seeds are kept in a standardized laboratory with room temperature of 25 +/-2 ℃, relative humidity of 50 +/-5 percent and 12h of light/12 h of dark.

2 method of experiment

2.1 Experimental animal grouping and handling

30 BABL/c mice were randomly divided into PBS normal group, OVA asthma model group, and (OVA + 50. mu.g PepN) treatment group, 10 mice each, with an experimental period of 22 days. The animal experiment molding method comprises the following steps: on days 0 and 7, two groups of mice except the normal group are sensitized by injecting 100 mu g of Ovalbumin (OVA) sensitizing solution into the abdominal cavity, and the mice in the normal group are injected with PBS into the abdominal cavity; two groups of mice, except the normal group, were challenged with 5% OVA by nebulization daily and the normal group mice were challenged with PBS by nebulization on days 14-21. The mice in the treatment group were treated by intraperitoneally anesthetizing with 1.5% sodium pentobarbital on days 2, 6, and 10 during the sensitization period of the mice, and then by instilling 50. mu.g of PepN through the nasal cavity.

2.2 counting of inflammatory cells in BALF

After the last excitation for 24h, the mice were anesthetized, endotracheal intubation was performed using an indwelling needle, the lungs were lavaged 5 times with 1ml of pre-cooled PBS with a recovery of not less than 90%, and the lavages were collected in clean Eppendorf tubes. Cells were harvested at 4 ℃ for 5min at 800g in a centrifuge, the cell pellet resuspended in 1ml PBS and the total number of inflammatory cells counted under the microscope. After smearing the cells by using a cell centrifugal smear machine, staining the cells by Reye, classifying and counting the proportion of various inflammatory cells under a microscope, and calculating the absolute value of the inflammatory cells.

2.3 Lung tissue section Observation

After the last excitation for 24h, the mice were anesthetized, the thoracic cavity was dissected, the lung tissue was excised, fixed in 4% paraformaldehyde solution, dehydrated, cleared, waxed, embedded, sectioned, and then HE-stained and PAS-stained, and finally the morphological changes of the tissue were observed under an optical microscope.

2.4 detection of IgE in serum

After 24h of the last challenge, the mice were anesthetized, the thoracic cavity was dissected, the mouse heart blood was collected using a 1ml syringe, after resting for 2h, centrifuged at 3000rpm for 15 min in a centrifuge, and the serum was collected in a clean centrifuge tube and frozen at-80 ℃. The content of IgE in mouse serum was determined according to the ELISA kit instructions.

2.5 detection of cytokines in BALF

After the last excitation for 24h, the mice were anesthetized, endotracheal intubation was performed using an indwelling needle, the lungs were lavaged 5 times with 1ml of pre-cooled PBS with a recovery of not less than 90%, and the lavages were collected in clean Eppendorf tubes. The supernatant was collected on a centrifuge at 4 ℃ for 5min at 800g and frozen at-80 ℃. Cytokines were measured in mouse BALF according to ELISA kit instructions.

3 results and analysis of the experiments

3.1 Effect of PepN on inflammatory cells in BALF in allergic asthma mice

Fig. 2A shows the counting result of total inflammatory cells in BALF of mice, and it can be seen from the figure that the total inflammatory cells in BALF of OVA asthma model group are significantly increased compared with those of PBS normal group, and the total inflammatory cells in BALF of allergic asthma mice are decreased after PepN treatment. Eosinophilia is one of typical characteristics of allergic asthma, and fig. 2B and 2C show that the proportion and total number of eosinophils in BALF of OVA asthma model group were significantly increased and the proportion and total number of eosinophils in BALF of allergic asthma mice were significantly decreased after PepN treatment, compared to PBS normal group mice.

3.2 Effect of PepN on Lung tissue morphology in mice with allergic asthma

Fig. 3A shows the observation result of HE staining of the lung tissue section of the mouse (magnification x 400), and it can be seen from the figure that, compared with the lung tissue of the normal group of mice, the pathological change of the lung tissue of the mice in the OVA model group is obvious, the normal structure of the pulmonary alveoli disappears, a large amount of inflammatory cells infiltrate around the bronchus, and the phenomena of airway lumen narrowing, wall deformation and the like occur, after PepN treatment, the inflammatory infiltration phenomenon of the lung tissue of the mice is obviously improved, and the alveolar space is complete. FIG. 3B shows the result of observing glycogen staining of mouse lung tissue sections (magnification. times.400), from which it can be seen that the production of goblet cells and mucus in lung tissues of mice in OVA model group is significantly increased compared to the lung tissues of normal group mice, and that the production of goblet cells and mucus in lung tissues of mice with allergic asthma is decreased after PepN treatment.

3.3 Effect of PepN on serum IgE in mice with allergic asthma

It is widely believed that the pathogenesis of allergic asthma is IgE-mediated hypersensitivity, namely IgE antibodies after contacting with specific antigens are combined with IgE receptors on the surfaces of mast cells and basophils, rapid degranulation is caused by bridging action, mediators which can cause local inflammation are released, and symptoms such as chest distress, wheezing, cough, breathlessness and the like are produced.

FIG. 4 shows the measurement results of the total IgE content in the serum of the mice, and it can be seen from the graph that the total IgE content in the serum of the mice of the OVA model group is obviously increased compared with that of the normal group, while the total IgE content in the serum of the mice of the PepN treatment group is reduced compared with that of the model group.

3.4 Effect of PepN on cytokines in BALF in allergic asthmatic mice

Allergic asthma is a disease in which excessive Th2 type immunity causes the immune imbalance of Th1/Th 2. IL-5 and IL-13 play important roles in Th2 cell regulation. IL-5 causes airway eosinophil aggregation, and IL-13 promotes mucus production and smooth muscle contraction, thereby causing inflammation.

FIGS. 5A and 5B show IL-5 and IL-13 levels in mouse BALF, respectively, and it can be seen that IL-5 and IL-13 levels in OVA model group mouse BALF were significantly increased compared to normal group mouse lung tissue, while IL-5 and IL-13 levels in allergic asthma mouse BALF were decreased after PepN treatment.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

sequence listing

<110> Chongqing university of medical science

application of streptococcus pneumoniae protein PepN in resisting allergic asthma

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<170> SIPOSequenceListing 1.0

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Met Gln Ala Val Glu His Phe Ile Lys Gln Phe Val Pro Glu His Tyr

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Asp Leu Phe Leu Asp Leu Ser Arg Glu Thr Lys Thr Phe Ser Gly Lys

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Val Thr Ile Thr Gly Gln Ala Gln Ser Asp Arg Ile Ser Leu His Gln

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Ala Gly Gln Val Glu Leu Val Leu Ala Phe Ser Gly Lys Ile Thr Asp

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Asn Met Thr Gly Ile Tyr Pro Ser Tyr Tyr Thr Val Asp Gly Val Lys

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Lys Glu Val Leu Ser Thr Gln Phe Glu Ser His Phe Ala Arg Glu Ala

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Phe Pro Cys Val Asp Glu Pro Glu Ala Lys Ala Thr Phe Asp Leu Ser

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Leu Arg Phe Asp Gln Ala Glu Gly Glu Leu Ala Leu Ser Asn Met Pro

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Glu Ile Asp Val Glu Asn Arg Lys Glu Thr Gly Ile Trp Lys Phe Glu

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Thr Thr Pro Arg Met Ser Ser Tyr Leu Leu Ala Phe Val Ala Gly Asp

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Leu Gln Gly Val Thr Ala Lys Thr Lys Asn Gly Thr Leu Val Gly Val

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Tyr Ser Thr Lys Ala His Pro Leu Ser Asn Leu Asp Phe Ser Leu Asp

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Tyr Pro Ile Pro Gln Ser Leu His Ile Ala Leu Pro Asp Phe Ser Ala

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Gly Ala Met Glu Asn Trp Gly Leu Val Thr Tyr Arg Glu Val Tyr Leu

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Val Val Asp Glu Asn Ser Thr Phe Ala Ser Arg Gln Gln Val Ala Leu

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Val Val Ala His Glu Leu Ala His Gln Trp Phe Gly Asn Leu Val Thr

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Met Lys Trp Trp Asp Asp Leu Trp Leu Asn Glu Ser Phe Ala Asn Met

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Met Glu Tyr Val Cys Val Asp Thr Ile Glu Pro Ser Trp Asn Ile Phe

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Glu Asp Phe Gln Thr Gly Gly Val Pro Leu Ala Leu Glu Arg Asp Ala

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

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Ile Asn Thr Leu Phe Asp Gly Ala Ile Val Tyr Ala Lys Gly Ser Arg

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Leu Met His Met Leu Arg Arg Trp Leu Gly Asp Ala Asp Phe Ala Lys

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Gly Leu His Ala Tyr Phe Glu Lys His Gln Tyr Ser Asn Thr Ile Gly

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Ser Asp Leu Trp Asp Ala Leu Gly Gln Ala Ser Gly Arg Asp Val Ala

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Ala Phe Met Asp Ser Trp Leu Glu Gln Pro Gly Tyr Pro Val Leu Thr

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Val Lys Val Glu Asn Asp Val Leu Lys Ile Ser Gln Lys Gln Phe Phe

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Ser Asn Trp Lys Gly Leu Pro Asp Thr Leu Glu Thr Glu Ser Ile Glu

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Leu Asn Thr Glu Asn Thr Ala His Tyr Ile Thr Asp Tyr Gln Gly Asp

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Leu Leu Glu Ala Val Leu Ala Glu Leu Glu Thr Leu Asp Asn Thr Ser

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Lys Leu Gln Ile Val Gln Glu Arg Arg Leu Leu Ala Glu Ala Gly His

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Ile Ser Tyr Ala Asp Leu Leu Pro Val Leu Asp Lys Leu Ala Lys Glu

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Glu Ser Tyr Leu Val Val Ser Ala Val Ser Gln Val Ile Ser Ala Leu

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Glu Arg Phe Ile Asp Glu Gly Thr Asp Ala Glu Thr Ala Phe Lys Gly

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Leu Val Ala Lys Leu Ala Arg His Asn Tyr Asp Arg Leu Gly Phe Glu

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Ala Lys Asp Gly Glu Ser Asp Glu Asp Glu Leu Val Arg Gln Leu Ala

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Val Ser Met Met Ile Arg Ser Asn Asp Ala Glu Ala Ser Gln Val Ala

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Ser Gln Ile Phe Ala Thr His Lys Glu Asn Leu Ala Gly Leu Pro Ala

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Ala Ile Arg Ser Gln Val Leu Ile Asn Glu Met Lys His His Glu Thr

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Lys Asp Leu Leu Ala Leu Tyr Leu Asp Thr Tyr Thr His Ala Thr Asp

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Ala Val Phe Lys Arg Gln Leu Ala Ala Ala Leu Ala Tyr Ser Thr Asp

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Ala Asp Asn Ile Gln Asn Leu Ile Thr Ser Trp Lys Asp Lys Phe Val

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Val Lys Pro Gln Asp Leu Ser Ala Trp Tyr Tyr Gln Phe Leu Ala His

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Gln Ala Thr Gln Lys Thr Ala Trp Ser Trp Ala Arg Glu Asn Trp Ala

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Trp Ile Lys Ala Ala Leu Gly Gly Asp Met Ser Phe Asp Ser Phe Val

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Ile Leu Pro Ala His Val Phe Lys Thr Gln Gln Arg Leu Ala Glu Tyr

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Lys Glu Phe Phe Glu Pro Gln Leu Ser Asp Leu Ala Leu Ser Arg Asn

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Ile Gly Met Gly Ile Lys Glu Ile Thr Ala Arg Val Asp Leu Ile Ser

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Arg Glu Lys Ala Ala Val Glu Ala Val Val Leu Gln Tyr Gly Asn Ala

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