Protein polypeptide and protein for use of marker in clinical diagnosis of non-tuberculosis mycobacterium infection and use thereof

Abstract

The application discloses a protein polypeptide for marker use in clinical diagnosis of non-tuberculous mycobacterial infection, comprising an amino acid sequence as shown in SEQ NO.1 or an amino acid sequence as shown in SEQ NO.2. The application also discloses proteins and uses thereof. The protein polypeptide of the application can specifically stimulate lymphocytes of a mycobacterium abscessus infected patient to secrete cytokines such as IFN-gamma, and therefore, the protein polypeptide can be used for marker use in clinical diagnosis of the mycobacterium abscessus infected patient.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a protein polypeptide and its protein and use for the use of markers in the clinical diagnosis of nontuberculosis mycobacterium infection. BACKGROUND

[0002] Nontuberculosis mycobacterium (NTM) refers to mycobacterium other than Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium leprae. The main NTM pathogenic bacteria clinically include Mycobacterium abscessus, etc. (1). Nontuberculosis mycobacterium disease refers to the lesions of related tissues or organs caused by human infection with NTM. In recent years, the prevalence rate of nontuberculosis mycobacterium lung disease has shown an increasing trend worldwide, and has become a common disease, which has posed a threat to human health (2).

[0003] Nontuberculosis mycobacterium (NTM) mainly involves the lung, and NTM lung disease lacks specific clinical manifestations and signs, which is difficult to differentiate from other diseases (3). Many clinicians often ignore the existence of NTM lung disease, and misdiagnosis or missed diagnosis often occurs. There is no efficient diagnosis method for NTM infection at present.

[0004] NTM infection can induce some immune cells of the body to be in an immune memory state. When these cells are stimulated again by NTM antigens, they will specifically produce a large amount of cytokines including interferon gamma (IFN-γ) (4), according to which it can be diagnosed whether the body is infected with NTM pathogenic bacteria. In the present application, the inventors screened two protein polypeptides that can be used for the diagnosis of Mycobacterium abscessus infection, and confirmed that the two polypeptides and their proteins have the potential to be used as markers in the clinical diagnosis of Mycobacterium abscessus infection.

[0005] REFERENCES

[0006] 1. Cara D Varley, Kevin L Winthrop, Nontuberculous Mycobacteria: Diagnosis and Therapy. Clin Chest Med, 2022 Mar; 43(1): 89-98. doi: 10.1016 / j.ccm.2021.11.007.

[0007] 2. Li-Ping Cheng, Shan-Hao Chen, Hai Lou, Xu-Wei Gui, Xiao-Na Shen, Jie Cao, Wei Sha, Qin Sun, Factors Associated with Treatment Outcome in Patients with Nontuberculous Mycobacterial Pulmonary Disease: A Large Population-Based Retrospective Cohort Study in Shanghai, Trop Med Infect Dis. 2022 Feb 15;7(2):27. doi: 10.3390 / tropicalmed7020027.

[0008] 3. Ye Gu, Chunyan Wu, Fangyou Yu, Xuwei Gui, Jun Ma, Liping Cheng, Qin Sun, Wei Sha, Application of endobronchial ultrasonography using a guide sheath and electromagnetic navigation bronchoscopy in the diagnosis of atypical bacteriologically-negative pulmonary tuberculosis, Ann Transl Med. 2019 Oct;7(20):567. doi: 10.21037 / atm.2019.09.37.

[0009] 4. Chi Yang, Xuejiao Luo, Lin Fan, Wei Sha, Heping Xiao, Haiyan Cui, Performance of Interferon-Gamma Release Assays in the Diagnosis of Nontuberculous Mycobacterial Diseases - A Retrospective Survey From 2011 to 2019, Front Cell Infect Microbiol. 2021 Feb 18;10:571230. doi: 10.3389 / fcimb.2020.571230. eCollection 2020.

[0010] 5. Rui Yang, Lan Yao, Ling Shen, Wei Sha, Robert L Modlin, Hongbo Shen, Zheng W Chen, IL-12 Expands and Differentiates Human Vγ2Vδ2 T Effector Cells Producing Antimicrobial Cytokines and Inhibiting Intracellular Mycobacterial Growth. Front Immunol. 2019 Apr 26; 10:913. doi: 10.3389 / fimmu.2019.00913. eCollection 2019. SUMMARY

[0011] To overcome the above-mentioned defects of the prior art, the purpose of the present application is to provide a protein polypeptide for use of a marker in clinical diagnosis of non-tuberculous mycobacterial infection.

[0012] In order to achieve the purpose of the present application, the technical solution adopted is:

[0013] The protein polypeptide for use of a marker in clinical diagnosis of non-tuberculous mycobacterial infection comprises:

[0014] The amino acid sequence as shown in SEQ NO. 1, the SEQ NO. 1 is as follows:

[0015] MSTDASGLGSPLETNLPLFVYGNLKPGELGHLLISPWVSDSRPATVTGHLWVRDGVPLADLGSRGHIRGHLLTLSAPGYRAVGELEPTAYYQWAKVTCIEPSRLKANTLVAAGWLTPDRGGGDVLYEPWTSTQDPLLTYGLAAVTDTLRNDGRAAFQGGQALYEPVHWLRFYRLQAAYMLACSILERIAFRLAPNAGPTTKVNILGRQPQFMSAVQSAGVPIPRRAVYRADNPRERVNLNKADQFANWAYQIRSNLVHRGKSASLEAELVRTALIDLHDVLRIYLQAAIPSISDTWMHADPTDSIRDWRIKTEFNAPPDN;

[0016] Or the amino acid sequence as shown in SEQ NO. 2, the SEQ NO. 2 is as follows:

[0017]

[0018] A protein constructed by at least one protein polypeptide used in the marker use in the clinical diagnosis of non-tuberculous mycobacterial infection.

[0019] Use of the protein polypeptide used in the marker use in the clinical diagnosis of non-tuberculous mycobacterial infection, which is used in the marker use in the clinical diagnosis of Mycobacterium abscessus infection.

[0020] The present application has the following beneficial effects:

[0021] The protein polypeptide of the present application can specifically stimulate the lymphocytes of the patients infected with Mycobacterium abscessus to secrete cytokines such as IFN-γ, and therefore they can be used in the marker use in the clinical diagnosis of the patients infected with Mycobacterium abscessus. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A schematic diagram of the ELISA of the present application for detecting the content of IFN-γ in cell culture supernatant.

[0023] Figure 2 A schematic diagram of the ELSPOT of the present application for detecting the number of IFN-γ secreting cells. DETAILED DESCRIPTION

[0024] The inventors performed proteomic analysis on the lung pathological tissues of the patients infected with Mycobacterium abscessus, and identified a plurality of protein polypeptides of Mycobacterium abscessus which may have subsequent application potential.

[0025] Through detection by enzyme linked immunosorbent assay (ELISA) and enzyme linked immunospot assay (ELISPOT), it was found that two polypeptides and their proteins can specifically stimulate the peripheral lymphocytes of the patients infected with Mycobacterium abscessus to produce IFN-γ. The two polypeptides and their proteins can be used in the marker use in the clinical diagnosis of Mycobacterium abscessus infection.

[0026] 1. Finding protein polypeptides of Mycobacterium abscessus in pathological tissues:

[0027] The lung lesion tissues of the patients infected with Mycobacterium abscessus were collected by surgical resection. After the tissues were lysed, proteomic analysis was performed, and a plurality of protein polypeptides of Mycobacterium abscessus were identified by comparison with a database.

[0028] The polypeptides were synthesized artificially, dissolved with sterilized water, and stored at -20℃ for standby.

[0029] The protein polypeptide sequence was subjected to BLAST analysis to obtain the complete protein sequence information containing the corresponding polypeptide. According to the protein sequence information, the complete protein was synthesized. After dissolving with sterilized water, it was stored at -20°C for standby.

[0030] 2. Preparation of peripheral blood PBMC cells:

[0031] Mycobacterium abscessus infected patients and healthy control population were recruited. 5ml of peripheral blood of the subjects was collected into an anticoagulant blood collection tube containing heparin sodium or sodium citrate, the blood collection tube was inverted and mixed thoroughly. According to the technology system we have established (as in reference 5), peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood by Ficoll-hypaque (polysucrose- metrizamide) density gradient centrifugation.

[0032] 3. ELISA detection:

[0033] 3.1 Stimulation of PBMC cells prepared in 2 with the protein polypeptide obtained in 1

[0034] (1) The prepared PBMC cells were plated in a 96-well plate, 100ul per well, 4 wells per sample for each sample.

[0035] (2) Each sample was added with stimulants according to the negative control well (cell culture medium), positive control well (phorbol ester PMA working concentration 100ng / ml), 417th polypeptide stimulation well (working concentration 1mg / ml), 421th polypeptide stimulation well (working concentration 1mg / ml), i.e. the protein polypeptide obtained in 1.

[0036] (3) After incubation of the cells for 20-24h, the cell culture supernatant was collected for subsequent ELISA detection.

[0037] 3.2 ELISA detection

[0038] (1) According to the number of experimental wells (blank and standard), determine the number of ELISA plate strips required. Samples (including standards) and blanks should be duplicated.

[0039] (2) Add sample: 100 L / well of diluted Cytokine standard (IFN-γ) to standard wells, 100 L / well of sample to sample wells, 100 L / well of Dilution buffer R (1x) to blank wells. Add sample: 100 mL / well of diluted Cytokine standard to standard wells, 100 mL / well of sample to sample wells, 100 mL / well of Dilution buffer R (1x) to blank wells.

[0040] (3) Add detection antibody: 50 L / well of Biotinylated antibody. Mix well and cover the plate with a sealing film. Incubate at room temperature (18-25°C) for 2 hours.

[0041] (4) Wash plate: remove the liquid in the wells, 300 L / well of 1x Washing buffer. Keep for 1 minute and then discard the liquid in the wells. Repeat 3 times, and dry on filter paper each time.

[0042] (5) Add enzyme: 100 L / well of Streptavidin-HRP. Cover the plate with a sealing film, and incubate at room temperature (18-25°C) for 20 minutes.

[0043] (6) Wash plate: repeat step (4).

[0044] (7) Color development: 100 mL / well of TMB (3,3',5,5'-tetramethylbenzidine) color developing solution. Incubate at room temperature (18-25°C) for 5-30 minutes in the dark. Determine the termination of the reaction according to the color depth (dark blue) in the wells (the reaction is terminated immediately when dark blue appears). Usually, 10-20 minutes of color development can achieve good results.

[0045] (8) Termination of reaction: 100 mL / well of Stop solution is quickly added to terminate the reaction.

[0046] (9) Read plate: read the values within 10 minutes after termination using a detection wavelength of 450 nm. It is recommended to read the plate using dual wavelengths, i.e., detection wavelength 450 nm, reference wavelength or correction wavelength 610-630 nm.

[0047] 4. ELISPOT detection:

[0048] 4.1 Stimulation of PBMC cells with protein polypeptides

[0049] (1) Take the PVDF plate pre-coated with anti-IFN-γ monoclonal antibody from 4°C and restore to room temperature. Add 200 μL RPMI-1640 medium to each well of the coated plate and let it stand at room temperature for 5-10 minutes, then remove the medium.

[0050] (2) Add stimulants: 10 μL / well, as follows:

[0051] Positive control well: add 50 ug / ml PHA (phytohemagglutinin);

[0052] Negative control well (including background blank control well): add RPMI-1640 medium;

[0053] Experimental well: add 20-100 ng / ml NTM synthetic protein.

[0054] (3) Add cell suspension: add the adjusted cell suspension to each experimental well, 100 μL / well.

[0055] (4) Incubate: after all samples and stimulants are added, cover the plate and place it in a 37°C, 5% CO2 incubator for 16-24 hours.

[0056] 4.2 ELISPOT detection:

[0057] (1) Pour out the cells and medium in the wells, add ice-cold deionized water, 200 μL / well, and place it in a 4°C refrigerator for 10 minutes to lyse the cells.

[0058] (2) Shake off the liquid in the wells, add 1x washing buffer, 260 μL / well, and let it stand for 1 minute, then discard the liquid in the wells and repeat six times, with each time being dried on a blotting paper.

[0059] (3) Incubate the antibody: add the diluted biotinylated antibody working solution to each experimental well, 100 μL / well. Incubate at 37°C for 1 hour.

[0060] (4) Repeat step (2).

[0061] (5) Add the diluted Streptavidin-HRP working solution to each experimental well, 100 μL / well. Incubate at 37°C for 1 hour.

[0062] (6) Discard the liquid in the wells, add 1 x Washing buffer, 260 μL / well, stay for 1 minute, discard the liquid in the wells, repeat five times, dry each time on the water-absorbing paper, then uncover the plate base, wash the membrane bottom and the base with deionized water / tap water, dry the residual water on the base and the membrane bottom with the water-absorbing paper, cover the base, add 1 x Washing buffer, 260 μL / well, stay for 1 minute, discard the liquid in the wells, and dry the liquid in the wells completely.

[0063] (7) Add the prepared AEC developing solution to each experimental well, 100 μL / well. Stay at room temperature for 5-30 minutes in the dark, and select the termination time of color development according to the generation of spots. If the room temperature is lower than 20°C, check every 5-10 minutes.

[0064] (8) Pour out the liquid in the wells, uncover the plate base, wash the front and back and the base with deionized water / tap water for 3-5 times to terminate the color development. Place the plate at room temperature in the shade, and dry naturally before covering the base.

[0065] (9) Count the spots on the ELISPOT plate with a magnifying glass or an inverted microscope. Each spot represents one IFN-γ secreting cell, i.e. spot forming units (SFU).

[0066] (10) The negative control well requires <10 SFU / 106 PBMC, and the positive control well requires >25 SFU / 106 PBMC.

[0067] 5. Results:

[0068] 5.1 Two protein polypeptides are screened to specifically stimulate the PBMC cells of NTM patients to secrete a large amount of IFN-γ.

[0069] In order to screen the polypeptides with potential clinical diagnostic application from the protein polypeptides obtained by mass spectrometric analysis, the inventors stimulated the PBMC cells from NTM patients and healthy control individuals with each polypeptide, respectively, and collected the culture supernatant.

[0070] The content of IFN-γ in the supernatant was detected by ELISA. The results showed that the content of IFN-γ in the culture supernatant of the PBMC cells of NTM patients stimulated by the polypeptides No. 417 and No. 421 (Table 1) was significantly higher than that of the unstimulated group Figure 1 ), i.e. the polypeptides No. 417 and No. 421 can specifically stimulate the PBMC cells of NTM patients to induce a large amount of IFN-γ.

[0071] In the present application, the polypeptides No. 417 and No. 421 are screened from the protein polypeptides obtained by mass spectrometric analysis of the NTM patient-derived PBMC cells, and the polypeptides No. 417 and No. 421 can specifically stimulate the PBMC cells of NTM patients to induce a large amount of IFN-γ. Figure 1Medium, PMA, No417 and No421 represent the number of IFN-γ positive cells in PBMC cells stimulated by medium, PMA, No417 and No421 respectively.

[0072] Table 1. Polypeptide information for potential clinical diagnosis of NTM infected patients.

[0073]

[0074]

[0075]

[0076] 5.2 Protein polypeptides No417 and No421 can specifically induce more PBMC cells of NTM patients to secrete IFN-γ.

[0077] In order to verify the specificity of the protein polypeptides to the PBMC cells from NTM, the inventors compared the number of IFN-γ secreting cells after stimulating the PBMC cells from NTM and healthy control (HC) with protein polypeptides by ELISPOT method.

[0078] The results showed that after stimulating with polypeptides No417 and No421, more IFN-γ positive spots were produced in the PBMC cells from NTM than in the PBMC cells from healthy control (HC) (as shown in Figure 2 ).

[0079] Figure 2 A represents the number of IFN-γ secreting positive cells in PBMC cells stimulated by polypeptide No417.

[0080] NTM represents the PBMC cells from NTM infected patients; HC represents the PBMC cells from healthy control. B represents the number of IFN-γ secreting positive cells in PBMC cells stimulated by polypeptide No421.

[0081] NTM represents the PBMC cells from NTM infected patients; HC represents the PBMC cells from healthy control.

[0082] Through the above experiments, the inventors of the present application unexpectedly found that:

[0083] Protein polypeptides No. 417 (sequence IDLHDVLRI) and No. 421 (sequence KGQIPVVI) of Mycobacteroides abscessus and their complete proteins (respectively: gamma-glutamyl cyclotransferase [Mycobacteroides abscessus], DNA-directed DNA polymerase III PolC [Mycobacteroides abscessus subsp. abscessus]) can specifically stimulate lymphocytes of patients infected with Mycobacteroides abscessus to secrete cytokines such as IFN-γ, so they can be used for the use of markers in the clinical diagnosis of patients infected with Mycobacteroides abscessus. SEQUENCE LISTING <110> Shanghai Pulmonary Hospital <120> Use of protein polypeptides and their proteins as markers in the clinical diagnosis of non-tuberculous mycobacterial infections and uses thereof <130> 20220613 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 320 <212> PRT <213> human sapiens <400> 1 Met Ser Thr Asp Ala Ser Gly Leu Gly Ser Pro Leu Glu Thr Asn Leu 1 5 10 15 Pro Leu Phe Val Tyr Gly Asn Leu Lys Pro Gly Glu Leu Gly His Leu 20 25 30 Leu Ile Ser Pro Trp Val Ser Asp Ser Arg Pro Ala Thr Val Thr Gly 35 40 45 His Leu Trp Val Arg Asp Gly Val Pro Leu Ala Asp Leu Gly Ser Arg 50 55 60 Gly His lie Arg Gly His Leu Leu Thr Leu Ser Ala Pro Gly Tyr Arg 65 70 75 80 Ala Val Gly Glu Leu Glu Pro Thr Ala Tyr Tyr Gin Trp Ala Lys Val 85 90 95 Thr Cys lie Glu Pro Ser Arg Leu Lys Ala Asn Thr Leu Val Ala Ala 100 105 110 Gly Trp Leu Thr Pro Asp Arg Gly Gly Gly Asp Val Leu Tyr Glu Pro 115 120 125 Trp Thr Ser Thr Gin Asp Pro Leu Leu Thr Tyr Gly Leu Ala Ala Val 130 135 140 Thr Asp Thr Leu Arg Asn Asp Gly Arg Ala Ala Phe Gin Gly Gly Gin 145 150 155 160 Ala Leu Tyr Glu Pro Val His Trp Leu Arg Phe Tyr Arg Leu Gin Ala 165 170 175 Ala Tyr Met Leu Ala Cys Ser lie Leu Glu Arg lie Ala Phe Arg Leu 180 185 190 Ala Pro Asn Ala Gly Pro Thr Thr Lys Val Asn lie Leu Gly Arg Gin 195 200 205 Pro Gin Phe Met Ser Ala Val Gin Ser Ala Gly Val Pro lie Pro Arg 210 215 220 Arg Ala Val Tyr Arg Ala Asp Asn Pro Arg Glu Arg Val Asn Leu Asn 225 230 235 240 Lys Ala Asp Gln Phe Ala Asn Trp Ala Tyr Gln Ile Arg Ser Asn Leu 245 250 255 Val His Arg Gly Lys Ser Ala Ser Leu Glu Ala Glu Leu Val Arg Thr 260 265 270 Ala Leu Ile Asp Leu His Asp Val Leu Arg Ile Tyr Leu Gln Ala Ala 275 280 285 Ile Pro Ser Ile Ser Asp Thr Trp Met His Ala Asp Pro Thr Asp Ser 290 295 300 Ile Arg Asp Trp Arg Ile Lys Thr Glu Phe Asn Ala Pro Pro Asp Asn 305 310 315 320 <210> 2 <211> 1033 <212> PRT <213> human sapiens <400> 2 Met Ile Ala Gln Leu Asp Thr Lys Thr Val Tyr Ser Phe Met Glu Ser 1 5 10 15 Met Val Ser Ile Lys Arg Tyr Val Ser Leu Gly Lys Glu Tyr Gly Tyr 20 25 30 Ser Ser Leu Gly Ile Met Asp Glu Asp Asn Leu Tyr Gly Ala Tyr Tyr 35 40 45 Phe Ile Lys Glu Cys Gln Lys Gln Gly Ile Gln Pro Leu Leu Gly Leu 50 55 60 Glu Ile Thr Val His His Lys Asp Glu Trp Ile Asn Leu Arg Phe Leu 65 70 75 80 Ala Leu Ser Asn Arg Gly Tyr Gln Asn Leu Met Lys Leu Ser Ser Leu 85 90 95 Lys Met Thr Gly Lys Lys Glu Trp Ala Asp Phe Ser Ser Tyr Leu Glu 100 105 110 Asp Ile Cys Val Ile Val Pro Tyr Tyr Ser Gly Met Asn Asp Leu Asp 115 120 125 Leu Gly His Asp Tyr Tyr Ile Gly Val Tyr Pro Asp Thr Pro Gln Ser 130 135 140 Asn Phe Ser His Pro Ile Leu Pro Leu Tyr Arg Val Asn Ser Phe Glu 145 150 155 160 Ser Glu Asp Leu Glu Ala Leu Gln Met Leu Lys Ala Ile Lys Glu Asn 165 170 175 Val Thr Leu Arg Glu Val Asp Val Gln Ser Gln Gln Gly Leu Phe Leu 180 185 190 Pro Ala Glu Arg Leu Glu Gln Phe Phe Leu Glu Lys Phe Pro Ile Ala 195 200 205 Leu Asp Asn Leu Ala Arg Leu Val Lys Asp Thr Ser Tyr Glu Ile Asp 210 215 220 Ser Ser Leu Lys Leu Pro Arg Phe Asn Pro Glu Arg Pro Ala Val Glu 225 230 235 240 Glu Leu Arg Glu Arg Ala Ile Lys Gly Leu Glu Glu Lys Gly Leu Leu 245 250 255 Asp Ser Val Tyr Gln Val Arg Leu Glu Glu Glu Leu Ser Val Ile His 260 265 270 Asp Met Gly Phe Asp Asp Tyr Phe Leu Val Val Trp Asp Leu Leu Arg 275 280 285 Phe Gly Arg Ser Gln Gly Tyr Tyr Met Gly Met Gly Arg Gly Ser Ala 290 295 300 Val Gly Ser Leu Val Ala Tyr Ala Leu Asp Ile Thr Gly Ile Asp Pro 305 310 315 320 Val Ala Lys Asn Leu Ile Phe Glu Arg Phe Leu Asn Arg Glu Arg Tyr 325 330 335 Thr Met Pro Asp Ile Asp Ile Asp Ile Pro Asp Ile Tyr Arg Pro Glu 340 345 350 Phe Ile Arg Tyr Val Arg Asp Arg Tyr Gly Ser Ile His Ala Ala Gln 355 360 365 Ile Val Thr Tyr Ser Thr Phe Gly Ala Lys Gin Ala He Arg Asp He 370 375 380 Phe Lys Arg Tyr Gly Val Pro Gin Tyr Gin Leu Thr Ala He Thr Lys 385 390 395 400 Lys He Gly Ser Lys Asp Thr Leu Thr Thr Ala Tyr Gin Gin Asn Leu 405 410 415 Gly Phe Arg Gin Leu He Gin Ser Lys He Gin Tyr Gin Lys Ala Phe 420 425 430 Ser He Ala Lys Lys He Gin Gly Tyr Pro Arg Gin Thr Ser He His 435 440 445 Ala Ala Gly Val Val He Ser Asp Lys Asn Leu Thr Asp Tyr He Pro 450 455 460 Leu Lys Tyr Gly Gin Asp Met Leu He Thr Gin Tyr Gin Ala Gin Gly 465 470 475 480 Val Gin Gly Asn Gly Leu Leu Lys Met Asp Phe Leu Gly Leu Arg Asn 485 490 495 Leu Thr Phe Ala Gin Lys Met Gin Gin Leu Leu Tyr Gin Thr Gin Gly 500 505 510 Ile Ser Leu Arg lie Glu Asp lie Asp Leu Glu Asp Lys Ala Thr Leu 515 520 525 Ala Leu Phe Ala Ala Gly Lys Thr Lys Gly lie Phe Gin Phe Glu Gin 530 535 540 Pro Gly Ala lie Arg Leu Leu Lys Arg Val Lys Pro Gin Asn Phe Glu 545 550 555 560 Glu Val Val Ala Thr Thr Ser Leu Asn Arg Pro Gly Ala Ser Asp Tyr 565 570 575 Ile Asp Asn Phe Val Ala Arg Lys His Gly Lys Glu Lys Val Thr Val 580 585 590 Leu Asp Pro Val Leu Glu Asp lie Leu Ala Pro Thr Tyr Gly lie Met 595 600 605 Leu Tyr Gin Glu Gin Val Met Gin Val Ala Gin Arg Tyr Ala Gly Phe 610 615 620 Ser Leu Gly Lys Ala Asp Met Leu Arg Arg Ala Met Gly Lys Lys Asn 625 630 635 640 Ala Ala Glu Met His Arg Met Glu Glu Ser Phe lie Gin Gly Ala Leu 645 650 655 Glu Lys Gly His Gly Gin Lys Gin Ala Gin Glu Val Phe Ala Val Met 660 665 670 Glu Lys Phe Ala Gly Tyr Gly Phe Asn Arg Ser His Ala Tyr Ala Tyr 675 680 685 Ala Ala Leu Ala Phe Gln Leu Ala Tyr Phe Lys Thr His Phe Pro Ala 690 695 700 Ile Phe Tyr Gln Val Met Leu Asn Tyr Ala Ser Gly Asp Tyr Ile Leu 705 710 715 720 Asp Ala Leu Glu Met Gly Phe Glu Leu Ala Pro Leu Ser Ile Asn Thr 725 730 735 Val Pro Tyr Gln Asp Lys Leu Ala Asp Lys Thr Ile His Leu Gly Leu 740 745 750 Lys Thr Ile Lys Gly Met Pro Arg Asp Phe Ala Tyr Trp Ile Ile Glu 755 760 765 His Arg Pro Phe Ser Ser Val Glu Asp Phe Ile Thr Arg Leu Pro Lys 770 775 780 Asn Tyr Gln Lys Ile Ser Leu Leu Thr Pro Leu Val Glu Val Gly Leu 785 790 795 800 Phe Asp Gly Phe Asp Lys Asn Arg Gln Lys Ile Leu Thr Asn Leu Pro 805 810 815 Thr Leu Phe Ile Phe Val Glu Glu Leu Gly Ser Leu Phe Ala Asp Ser 820 825 830 Ser Tyr Ser Trp Thr Asp Thr Glu Asp Phe Ser Asn Ile Glu Lys Phe 835 840 845 Gln Lys Glu Gln Glu Trp Leu Gly Val Gly Ile Ser Gln His Pro Leu 850 855 860 Leu Val Leu Ala Lys Asn Pro Leu Tyr Pro Ile Val Ser Leu Ser Glu 865 870 875 880 Leu Ser Glu Gly Gln Thr Ala Thr Val Leu Val Glu Ile Gln Ser Ile 885 890 895 Arg Val Ile Arg Thr Lys Lys Gly Glu Asn Met Ala Phe Leu Lys Val 900 905 910 Thr Asp Ser Lys Thr Ser Leu Glu Val Thr Val Phe Ser Glu Gln Tyr 915 920 925 Arg Gln Phe Lys Asn Leu Leu His Glu Gly Ser Phe Tyr Tyr Leu Asn 930 935 940 Gly Lys Val Gln Ala Arg Asp Gly Arg Leu Gln Leu Val Leu Asn Asn 945 950 955 960 Leu Lys Glu Ala Val Ser Glu Arg Phe Trp Ile Gln Val Pro Asn His 965 970 975 Asp His Asp Ser Glu Ile Tyr His Ile Leu Asp Gln Tyr Lys Gly Gln 980 985 990 Ile Pro Val Val Ile Arg Tyr Glu Asn Glu Gin Lys Asn Ile Leu Leu 995 1000 1005 Pro Gly Tyr Leu Val Ala Lys Asp Ala Gly Leu Gin Gin Ser Leu Lys 1010 1015 1020 Gln Ile Val Met Lys Thr Ile Tyr Arg 1025 1030

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