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CN1518595A - modified arginine deiminase - Google Patents

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CN1518595A
CN1518595A CNA01819673XA CN01819673A CN1518595A CN 1518595 A CN1518595 A CN 1518595A CN A01819673X A CNA01819673X A CN A01819673XA CN 01819673 A CN01819673 A CN 01819673A CN 1518595 A CN1518595 A CN 1518595A
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迈克・A・克拉克
迈克·A·克拉克
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Phoenix Pharmacologics Inc
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Abstract

The present invention is directed to arginine deiminase modified with polythylene glycol, to methods of treating cancer, and to methods of treating and(or inhibiting metastasis.

Description

The arginine deiminase of modifying
Related application
The application is U.S. Patent application No.09/023, and 809 part continuation application requires the U.S. Provisional Patent Application No.60/046 that submitted on May 12nd, 1997,200 right of priority thus.
Invention field
The present invention relates to method through polyethyleneglycol modified arginine deiminase, treatment method for cancer and treatment and/or inhibition transitivity illness.
Background of invention
Malignant melanoma (stage 3) and liver cancer are the mortality diseases of killing Most patients in a year diagnosing.Annual nearly 16,000 people of the U.S. die from these diseases.Just promptly increase at the melanomatous sickness rate of the U.S., in other countries, as Australia's its rate of rise even higher.The place of hepatitis epidemic in the world, the sickness rate of liver cancer is higher, and for example, in Japan and Taiwan, liver cancer is one of primary cancer.Urgent need is effectively treated these diseases.
Have and treat some cancer with the method for selective removal indispensable amino acid.The most famous example is to utilize the altheine enzyme to reduce the level of l-asparagine to treat acute lymphocytoblast leukemia.The most frequently used altheine enzyme separates from intestinal bacteria.But because making in clinical application, this enzyme institute inherent antigenicity and short circulating half-life be restricted, referring to Y.K.Park etc., and Anticancer Res., 1:373-376 (1981).Bacillus coli L-asparaginase enzyme antigenicity with the polyoxyethylene glycol covalent modification is minimized, and circulating half-life is prolonged, referring to Park, and Anticancer Res., the same; Y.Kamisald etc., J Pharmacol.Exp.Ther., 216:410-414 (1981); With Y.Kamisaki etc., Gann., 73:47-474 (1982).Attempting to identify the indispensable amino acid degrading enzyme that other are used for the treatment of cancer although paid many effort, do not succeed, mainly is because produce many severe side effect by limiting the removal indispensable amino acid.
Existing degraded non-essential amino acid such as the arginic enzyme of reporting may be the effective ways of some cancer of control.For example, isolating arginine deiminase (ADI) from pseudomonas putida (Pseudomonas pudita), referring to J.B.Jones, " arginine deiminase is to the lymphoblastic influence of murine leukemia (The Effect of Arginine Deiminase on MurineLeukemic Lymphoblasts) ", Ph D dissertation, The University of Oklahoma, 1-165 page or leaf (1981).Although can be at the external cell of kill tumor effectively, but by the effect of failing to demonstrate in vivo of isolating ADI in the pseudomonas putida (P.pudita), this is because it shows enzymic activity hardly under neutral pH, and is promptly removed from the blood circulation of laboratory animal.Arginine deiminase from mycoplasma arginini (Mycoplasmaarginini) has been described, referring to for example Takaku etc., Int.J.Cancer in the following document, 51:244-249 (1992) and U.S. Patent No. 5,474,928, its disclosed content is hereby incorporated by.Yet the therepic use of this heterologous protein relates to antigenic problem.Also have and describe to claim to utilize polyoxyethylene glycol to pass through the cyanuryl chloride linking group arginine deiminase of mycoplasma arginini (Mycoplasme arginini) is carried out chemically modified, referring to Takaku etc., Jpn.J Cancer Res., 84:1195-1200 (1993).But owing to discharge prussiate from the cyanuryl chloride linking group, the albumen after the above-mentioned modification is deleterious in metabolism.
Need to degrade non-essential amino acid at present, do not produce the composition of the problem that is associated with prior art again.The present invention relates to these and other free-revving engine.
Summary of the invention
The present invention relates to through polyethyleneglycol modified arginine deiminase.In preferred embodiments, this arginine deiminase is that about 1,000 to about 50,000 polyoxyethylene glycol is directly or by a kind of biocompatibility linking group modification through total weight-average molecular weight.
Another embodiment of the present invention relates to the treatment method for cancer, and described cancer comprises for example sarcoma, liver cancer and melanoma.The invention still further relates to treatment and/or suppress the method that tumour cell shifts.
These and other aspect of the present invention will partly be illustrated in following detailed Description Of The Invention.
Brief description of the drawings
Fig. 1 represents the aminoacid sequence of arginine deiminase, described aminoacid sequence clone is from mycoplasma arginini (Mycoplasma arginini) (the aminoacid sequence SEQ IDNO:1 on top, be called ADIPROT), mycoplasma arthritidis (Mycoplasma arthritides) (the aminoacid sequence SEQ ID NO:2 at middle part, be called ARTADIPRO) and mycoplasma hominis (Mycoplasma hominus) (the aminoacid sequence SEQ ID NO:3 of bottom is called HOMADIPRO).
Fig. 2 A and 2B represent that the single dose natural arginine takes off the imines enzyme and the arginine deiminase modified through polyoxyethylene glycol (as molecular weight 5,000) to the effect of mice serum arginine-level and serum citrulline level.
Fig. 3 represents to work as PEG10,000 by different linking groups and ADI when covalently bound to the influence of serum arginine-level.
When Fig. 4 represented with the PEG-ADI injection mouse of single dose, the molecular weight of linking group and polyoxyethylene glycol was to the wherein influence of citrulline output.
Fig. 5 A and 5B represent by ADI-SS-PEG5, the dose response of 000 serum arginine that causes and citrulline level.Fig. 5 C and 5D represent by ADI-SS-PEG20, the dose response of 000 serum arginine that causes and citrulline level.
Fig. 6 represents natural A DI, ADI-SS-PEG5,000 and ADI-SS-PEG20, and 000 antigenicity.
Fig. 7 represents to use ADI-SS-PEG5, and 000, ADI-SS-PEG12,000 or ADI-SS-PEG20,000 treatment is to the influence of the tumour size in the mouse of having injected SK-mel 2 human melanoma cells.
Fig. 8 represents to use ADI-PEG20, and 000 treatment is to the influence of the tumour size in the mouse of having injected SK-mel 28, SK-mel 2 or M24-met human melanoma cell.
Fig. 9 represents to use ADI-PEG5, and 000, ADI-PEG12,000 or ADI-PEG20,000 treatment is to the influence of the mouse survival of having injected people's liver cancer SK Hep1 cell.
Figure 10 represents the aminoacid sequence of arginine deiminase, described aminoacid sequence clone is from streptococcus pyogenes (Steptococcus pyogenes) (the aminoacid sequence SEQ IDNO:6 on top, be called STRADIPYR) and streptococcus pneumoniae (Steptococcus pneumoniae) (the aminoacid sequence SEQ ID NO:7 of bottom is called STRADIPNE).
Figure 11 represents the aminoacid sequence of arginine deiminase, described aminoacid sequence clone is from B. burgdorferi (Borrelia burgdorferi) (the aminoacid sequence SEQ IDNO:8 on top, be called BORADIBUR) and Ah's borrelia burgdorferi (Borrelia afzelii) (the aminoacid sequence SEQ ID NO:9 of bottom is called BORADIAFZ).
Figure 12 represents aminoacid sequence (the aminoacid sequence SEQ ID NO:10 on top of Qiardiu intestinalis, be called QIAADIINT), aminoacid sequence (the aminoacid sequence SEQ ID NO:11 at middle part of bacillus aerogenes capsulatus (Clostridiumperfringens), be called CLOADIPER) and the aminoacid sequence (the aminoacid sequence SEQ ID NO:12 of bottom is called BACADILIC) of Bacillus licheniformis (Bacillus licheniformis).
Figure 13 represents enterococcus faecalis (Enterococcus faecalis) (the aminoacid sequence SEQ ID NO:13 on top, be called ENTADIFAE) and the aminoacid sequence (the aminoacid sequence SEQ ID NO:14 of bottom is called LACADISAK) of pure mellow wine breast genus bacillus (Lactobacillussake).
Detailed Description Of The Invention
Normocellular growth does not need arginine, and this is because they can synthesize arginine from citrulling by argininosuccinate synthetase and the reaction of argininosuccinase catalyse two-step. On the contrary, melanoma, hepatoma and some sarcomas are not expressed argininosuccinate synthetase; So they are arginine auxotroph. This Difference of Metabolism can be used to exploitation to treating the safe and effective therapeutic agent of these cancers. Arginine deiminase catalysis arginine transforms to citrulling, can be used for removing arginine. Therefore, arginine deiminase can be used for treating melanoma, hepatoma and some sarcomas.
Be present in natural arginine in the microorganism to take off the imines enzyme be antigenic and can promptly from patient's blood circulation, be removed. These problems can be overcome by the arginine deiminase through polyethylene glycol (PEG) covalent modification. Through the arginine deiminase of polyethylene glycol covalent modification (have or do not have linking group) hereinafter to be referred as " ADI-PEG ". Take off the imines enzyme with natural arginine and compare, ADI-PEG has kept the antigenicity of its most enzymatic activity, much less, has greatly prolonged circulating half-life, and more effective aspect the treatment tumour.
" polyethylene glycol " or " PEG " refers to the mixture of the condensation polymer of the oxirane of side chain or linear form and water, by general formula H (OCH2CH 2) nOH represents that wherein n is at least 4. " polyethylene glycol " or " PEG " occurs with the numeric suffix of its approximate mean molecule quantity of expression. For example, PEG5, the total weight average molecular weight of 000 finger is about 5,000 polyethylene glycol; PEG12, the total weight average molecular weight of 000 finger is about 12,000 polyethylene glycol; And PEG20, the total weight average molecular weight of 000 finger is about 20,000 polyethylene glycol.
" melanoma " may be a kind of by skin and other organs, comprises that the melanophore system of oral cavity, oesophagus, anal tube, vagina, pia arachnoid and/or conjunctiva or eyes develops and next pernicious or innocent tumour.Term " melanoma " for example comprises melanoma and surperficial propagated melanoma under acra-freckle (acral-lentiginous) melanoma, amelanotic melanoma, optimum juvenile form (juvenile) melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, the nail.
" hepatoma " may be the pernicious or innocent tumour of a kind of liver, comprises for example hepatocellular carcinoma.
" patient " refers to animal, preferred mammal, more preferably people.
" biocompatibility " is often referred to such material or compound, and they do not damage biological function, do not produce the unacceptable toxicity of any degree, comprise allergy and morbid state.
Use following abbreviated form in this specification sheets: PEG, polyoxyethylene glycol; ADI, arginine deiminase; SS, the succsinic acid succinimide ester; SSA, the succinimido succinic diamide; SPA, the propionic acid succinimide ester; And NHS, N-hydroxy-succinamide.
The present invention is based on unexpected a discovery, promptly through polyethyleneglycol modified ADI the cancer and the inhibition metastasis of cancer for the treatment of some type is had significant effect.By or not by the situation of linking group under ADI can link to each other with the polyoxyethylene glycol covalency, utilized linking group in preferred embodiments.
In the present invention, the arginine deiminase gene can comprise as deriving, clone or produce in microorganism, reorganization biotechnology or its arbitrary combination from any source.For example, arginine deiminase can be cloned from the microorganism of Mycoplasma (Mycoplasma), Clostridium (Clostridium), bacillus (Bacillus), Borrelia (Borrelia), enterococcus spp (Enterococcus), streptococcus (Streptococcus), lactobacillus (Lactobacillus), Qiardia and be obtained.Described arginine deiminase is preferably cloned from mycoplasma pneumoniae (Mycoplasma pneumoniae), mycoplasma hominis (Mycoplasma hom, inus), mycoplasma arginini (Mycoplasma arginini), streptococcus pyogenes (Steptococcuspyogenes), streptococcus pneumoniae (Steptococcus pneumoniae), B. burgdorferi (Borrelia burgdorferi), Ah's borrelia burgdorferi (Borrelia afzelii), Qiardiaintestinalis, bacillus aerogenes capsulatus (Clostridium perfringens), Bacillus licheniformis (Bacillus licheniformis), enterococcus faecalis (Enterococcus faecalis), pure mellow wine breast genus bacillus (Lactobacillus sake) or its arbitrary combination.Particularly, be used for arginine deiminase of the present invention and can have the one or more aminoacid sequences shown in Fig. 1 and the 10-13.
In particular of the present invention, preferably, described arginine deiminase clone is from the microorganism of Mycoplasma.More preferably, described arginine deiminase clone is from mycoplasma arginini (Mycoplasnia arginini), mycoplasma hominis (Mycoplasma hominus) and mycoplasma arthritidis (Mycoplasnia arthritides) or its arbitrary combination.Particularly, be used for arginine deiminase of the present invention and can have one or more aminoacid sequences shown in Figure 1.
In one embodiment of the invention, to have total weight-average molecular weight be about 1,000 to about 50,000 to described polyoxyethylene glycol (PEG); More preferably from about 3,000 to about 40,000, more preferably from about 5,000 to about 30,000; More preferably from about 8,000 to about 30,000; More preferably from about 11,000 to about 30,000; More preferably from about 12,000 to about 28,000; More preferably from about 16,000 to about 24,000; More preferably from about 18,000 to about 22,000; More preferably from about 19,000 to about 21,000 and most preferably from about 20,000.Usually, have 30,000 or more the high-molecular weight polyoxyethylene glycol be difficult to dissolving, cause preparation output to reduce greatly.Polyoxyethylene glycol can be side chain or straight chain, preferred straight chain.Usually, improve the immunogenicity of the molecular weight reduction ADI of polyoxyethylene glycol.Have the molecular weight polyethylene glycol described in the present embodiment can with ADI, and optional connection with a kind of biocompatible linking group, comprise for example melanoma, hepatoma and sarcoma, preferably melanoma to treat cancer.
In another embodiment of the invention, described polyoxyethylene glycol has total weight-average molecular weight about 1,000 to about 50,000; Preferred about 3,000 to about 30,000; More preferably from about 3,000 to about 20,000; More preferably from about 4,000 to about 12,000; More preferably from about 4,000 to about 10,000; More preferably from about 4,000 to about 8,000; More preferably from about 4,000 to about 6,000; Most preferably from about 5,000.Polyoxyethylene glycol can be side chain or straight chain, preferred straight chain.Have the molecular weight polyethylene glycol described in the present embodiment can with ADI, and optional connection with a kind of biocompatible linking group, comprise for example hepatoma, hepatoma and sarcoma, preferably hepatoma to treat cancer.
Being used for the covalently bound connection gene to PEG of ADI can be biocompatibility linking group arbitrarily.As mentioned above, " biocompatible " refers to those nontoxic can uses and do not cause damage, little illness, disease or dead compound or group in external or body.PEG can be by for example ehter bond, ester bond, mercaptan key or amido linkage link to each other with linking group.Suitable biocompatibility linking group comprises for example ester group, amide group, imide, carbamate groups, carboxyl, hydroxyl, carbohydrate, the succinimide group (comprises for example succsinic acid succinimide ester (SS), propionic acid succinimide ester (SPA), carboxymethylation succinimide (SCM), succinimido succinic diamide (SSA) or N-hydroxy-succinamide (NHS)), epoxide group, oxygen base carbonylic imidazole base (comprising for example carbonyl dimidazoles base (CDI)), nitrophenyl (comprising for example carbonic acid nitro phenyl ester (NPC) or carbonic acid trichlorine phenyl ester (TPC)), the trysylate group, aldehyde radical, isocyanate group, the ethene sulfuryl, the tyrosine group, the halfcystine group, Histidine group or primary amine.Preferably, described biocompatibility linking group is ester group and/or succinimido.More preferably, this linking group is SS, SPA, SCM, SSA or NHS; More preferably SS, SPA or NHS, most preferably SS or SPA.
Perhaps, ADI can the direct and PEG coupling (promptly not having linking group) by amino, sulfydryl, hydroxyl or carboxyl.
Utilize method well known in the prior art, ADI can be covalently attached to PEG by the biocompatibility linking group, referring to for example, Park etc., Anticancer Res., 1:373-376 (1981); And Zaplipsky and Lee, Polyethylene Glycol Chemistry:Biotechnical and Biomedical Applications, J.M.Harris compiles, Plenum Press, NY, Chapter 21 (1992), and its disclosed content is hereby incorporated by.
PEG is connected to the circulating half-life that can prolong ADI on the ADI.Usually, PEG links to each other with the primary amine of ADI.Known as those skilled in the art, on arginine deiminase, select the polyoxyethylene glycol connection site to depend on the effect in each site in this protein active structural domain.PEG can be connected on the primary amine of arginine deiminase under the prerequisite of not losing enzymic activity basically.For example, have about 17 Methionins that can modify through this program by the ADI that clones in mycoplasma arginini, mycoplasma arthritidis and the mycoplasma hominis.In other words, these 17 Methionins all are to make ADI pass through the possible site that the biocompatibility linking group links to each other with PEG, described biocompatibility linking group such as SS, SPA, SCM, SSA and/or NHS.PEG also can link to each other in other sites with ADI, content disclosed according to the present invention, and this is conspicuous to those skilled in the art.
Have 1 can link to each other with the ADI covalency to about 30 PEG molecules.Preferably, ADI is through about 7 to about 15 PEG molecular modifications, more preferably through about 9 to about 12 PEG molecular modifications.In other words, about 30% is modified by PEG to about 70% primary amino on the arginine deiminase, and preferred about 40% to about 60%, and more preferably from about 45% to about 55%, and most preferably about 50% primary amino is modified by PEG on the arginine deiminase.When terminal, preferably only utilized a PEG molecule to ADI when PEG is covalently bound.PEG number on the increase ADI can prolong the circulating half-life of this enzyme.Yet the PEG number on the increase ADI reduces the specific activity of this enzyme.Therefore, need between to reach balance, content disclosed according to the present invention, this is apparent to one skilled in the art.
Among the present invention, the common trait of most preferred biocompatibility linking group is that they all are to link to each other with the primary amine of arginine deiminase by maleimide groups.In case with the arginine deiminase coupling, SS-PEG has had the ester bond of contiguous PEG, this can make this site to the serum esterase sensitivity, thereby in vivo PEG is discharged from ADI.SPA-PEG and PEG2-NHS do not have ester bond, so they are insensitive to serum esterase.
In the present invention, as if specific linking group does not influence circulating half-life or its specific enzymes activity of PEG-ADI.Yet applying biological consistency linking group is extremely important in the present invention.The PEG that links to each other with albumen can be a strand, as SS-PEG, SPA-PEG and SC-PEG, also can use side chain PEG, as PEG2-NHS.The preferred following elaboration of linking group structural formula among the present invention.
Figure A0181967300151
The treatment significant quantity of one of compound of the present invention is to suppress the amount of tumor growth effectively.Usually, treatment begins with low dose, increases dosage then gradually until the best effect that reaches under the described environment.Usually, the therapeutic dose of compound of the present invention can be from about 1 to about 200mg/kg, and twice to approximately whenever biweekly weekly.For example, described dosage can be that about 1mg/kg is weekly, is equivalent to the 2ml intravenous injection, to per 3 days of about 20mg/kg once.ADI-SS-PEG5, the dosage of the best of 000 can be approximately weekly twice, and ADI-SS-PEG20,000 optimal dose can be from a week approximately once to per two weeks approximately once.Before the injection, PEG-ADI can mix mutually with phosphate buffered saline buffer or any suitable solution well known by persons skilled in the art.As required, described PEG-ADI preparation can solid (cold dry) or liquid dosage form use.
Method of the present invention comprises external or the interior application of body.With regard to external application, comprise cell cultures, compound described herein can be added in the cultured cells, then incubation.Use antibody producing technology well-known in the art, compound of the present invention also can be used for promoting mono-clonal and/or polyclonal antibody to produce.Above-mentioned mono-clonal and/or polyclonal antibody can be used for multiple diagnostic uses, and this also is conspicuous to those skilled in the art.
The approach of using compound of the present invention in vivo is different and different according to the purpose that will reach.As this field technicians can recognize, can use PEG-ADI composition of the present invention by following manner, in for example oral, the nose, in the intraperitoneal, parenteral, intravenously, intralymphatic, tumour, in the intramuscular, a matter, in the intra-arterial, subcutaneous, intraocular, synovial membrane, through epithelium with through skin.
Embodiment
Further specify the present invention by following embodiment, these embodiment have been the effect of explanation, scope of the present invention are not imposed any restrictions.
Embodiment 1: the generation of reorganization ADI
The culture of mycoplasma arginini (mycoplasnia arginini) (ATCC 23243), mycoplasma hominis (mycoplasma hominus) (ATCC 23114) and mycoplasma arthritidis (mycoplasma arthritides) (ATCC 23192) is from American type culture collection Rockville, and Maryland obtains.
From mycoplasma arginini (mycoplasnia arginini), mycoplasma hominis (Mycoplasmahominus) and mycoplasma arthritidis (mycoplasma arthritides), clone arginine deiminase and at expression in escherichia coli, referring to S.Misawa etc., J.Biotechnology, 36:145-155 (1994), disclosed content is incorporated herein by reference with it in full at this in the document.Fig. 1 has provided the aminoacid sequence from the arginine deiminase in above-mentioned each species.The aminoacid sequence on top is called ADIPROT and comes from mycoplasma arginini (mycoplasniaarginini); The intermediary aminoacid sequence is called ARTADIPRO and comes from mycoplasma arthritidis (mycoplasma arthritides), and the aminoacid sequence of bottom is called HOMADIPRO and comes from mycoplasma hominis (Mycoplasma hominus).Each aminoacid sequence all has sequence conservative more than 96%.Characterize each proteinic specific enzymes activity, K with method known to those skilled in the art m, V MaxWith best pH, show that they are undistinguishable to each other aspect biological chemistry.Determine best pH with citrate buffer solution (pH 5-6.5), phosphoric acid buffer (pH 6.5-7.5) and borate buffer (pH 7.5-8.5).Make enzyme with the arginine incubation of different concns and measure citrulline output, determine K thus mAnd V MaxThe K of different enzymes mBe about 0.02 to 0.06 μ M, and V MaxBe mol/ minute/mg of about 15-20 μ, these are worth all in standard error scope separately.
By the primer that is derived from oneself disclosed mycoplasma arginini sequence to by PCR reaction amplification arginine deiminase gene, referring to for example T.Ohno etc., Ihfect.Immun., 58:3788-3795 (1990), disclosed content is incorporated herein by reference with it in full at this in the document:
SEQ?ID?NO：4，5′-GGGATCCATGTCTGTATTTGACAGT-3′
SEQ ID NO:5,5 '-TGAAAGCTTTTACTACCACTTAACATCTTTACG-3 ' is cloned into the product of PCR reaction among the expression plasmid pQE16 with the segmental form of BamHI-HindIII.Dna sequence analysis show by pcr amplification go out from the fragment of mycoplasma arginini and Ohno etc., Infect.Immun., the same in disclosed arginine deiminase gene have same sequence.Before expression in escherichia coli, change the TGA codon of 5 coding colors propylhomoserins in the ADI gene into TGG by oligonucleotide-directed mutagenesis, at gene referring to J.R.Sayers etc., Biotechniques, 13:592-596 (1992).Reorganization ADI is with the formal representation of inclusion body, and described inclusion body accounts for 10% of total cell protein.
Albumen from each species in above-mentioned three mycoplasma species has 95% homology, and is easy to pass through column chromatography purification.From 1 liter of fermented liquid, can be purified into about 200mg pure protein.Reorganization ADI can stablize about fortnight down at 37 ℃, and can preserve 8 months at least under 4 ℃.The application method known to those skilled in the art determines this albumen arginine is had high affinity (0.04 μ M), and best physiological pH is about 7.2 to about 7.4.
Embodiment 2: renaturation and the purifying of reorganization ADI
Make the ADI protein renaturation that has less modification, referring to Misawa etc., J.Biotechnology, 36:145-155 (1994), disclosed content is incorporated herein by reference with it in full at this in the document.The 100g cytoplasm is resuspended in the 10mM K of 800ml 2PO 4, pH7.0, among the 1mM EDTA (damping fluid 1), cell micro-flowmeter (Microfluidizer) (Microfluidics Corporation, Newton, MA) in by twice so that cell rupture.Add Triton-100 and reach final concentration 4% (v/v).Described homogenate was stirred 30 minutes down at 4 ℃, then with 13, and centrifugal 30 minutes of 000g.Collecting precipitation is resuspended in it in 1 liter of damping fluid 1 that contains 0.5% TritonX-100.(Microgon Inc., Laguna Hills CA) filter above-mentioned solution thoroughly to the sex change damping fluid (50mM Tris HCl, pH 8.5,10mM DTT) of 5 times of volumes with the hollow fiber column with 100kD rejection.Add Guanidinium hydrochloride and reach final concentration 6M, described solution was stirred 15 minutes down at 4 ℃.Make gained solution in folding damping fluid 1 (10mM K again 2PO 4, pH7.0) in 100 times of dilutions, and stirred 48 hours down at 15 ℃, 15, the centrifugal removal granular substance of 000xg.
The supernatant liquor warp of gained is used the Q Sepharose FastFlow that folds the damping fluid balance again and cross in advance, and (Pharmacia Inc., Piscataway NJ) concentrates.With the folding again buffer solution elution ADI that contains 0.2M NaCl.Analyze through SDS-PAGE, the ADI lipidated protein that is generated by above-mentioned purge process is higher than 95%.From the 1kg cytoplasm, can be purified into the ADI albumen of the pure renaturation of 8g, be equivalent to the ADI of every liter of tunning 200mg purifying.
With Oginsky etc., Meth.Enzymol., little modification method of describing among (1957) 3:639-642 is measured the ADI activity.With 0.1M Na 2PO 4, 10 μ l samples among the pH 7.0 (BUN tests damping fluid) placed 96 hole titer plate, add the 0.5mM arginine in the 40 μ l BUN test damping fluid, cover culture plate, in 37 ℃ of following incubations 15 minutes.Add 20 μ l BUN reagent (Sigma Diagnostics) completely, with titer plate 100 ℃ of following incubations 10 minutes.Described titer plate is cooled to 22 ℃, and (Molecular Devices analyzes under 490nm Inc) with the titer plate readout instrument.Per minute is decided to be 1.0IU with the enzyme gauge that the L-arginine of 1 μ mol changes into L-guanidine propylhomoserin.(Pierce Co., Rockford IL), determine protein concentration with bovine serum albumin as standard with Pierce Coomassie blue protein analysis reagent.
The ADI goods enzymic activity of purifying is 17-25IU/mg.
Embodiment 3:PEG and ADI are connected
In the phosphoric acid buffer of 100mM, pH 7.4, PEG is linked to each other with the ADI covalency.In brief, the ADI in the phosphoric acid buffer mixes mutually with excessive 100mol PEG.At room temperature stirring reaction is 1 hour, then described mixture is dialysed to remove uncombined PEG.
First experiment is used for assessing the effect of linking group used in the PEG-ADI composition.PEG is that ester group or maleimide base comprise that SS, SSA, SPA and SSPA covalency link to each other with ADI by four kinds of different linking groups, and wherein total weight-average molecular weight of PEG is 5,000,10,000,12,000,20,000,30,000 and 40,000; Epoxy group(ing), epoxy-PEG wherein total weight-average molecular weight of PEG is 5,000; And branched PEG group, PEG2-NHS, wherein total weight-average molecular weight of PEG is 10,000,20,000 and 40,000.
The resulting composition of 5.0IU is expelled to (every group of each 5 mouse) in the mouse.In order to measure the arginine serum level, mouse is passed through eye socket blood vessel (retro orbital plexus) bloodletting (100 μ l).Add isopyknic 50% (w/v) trichoroacetic acid(TCA) after the collection immediately.Centrifugal (13,000xg, 30 minutes) remove precipitation, get supernatant in-70 ℃ of preservations.With full-automatic amino acidanalyser of Beckman Instruments and reagent, according to the program analysis sample that manufacturers provided.This method is about 2-6 μ M to arginic sensitivity limit, and the repeatability of measured value is in about 8%.Determine the arginic amount of serum by amino acid analysis.Shown in Fig. 3 result, the linking group of covalent bonding PEG and ADI reduces the arginic ability of serum to ADI does not in vivo have appreciable effect.In other words, must use the nontoxic linking group except that using in vivo, this linking group is very unimportant to experimental result.
The molecular weight that second experiment is used to estimate linking group and PEG is in vivo to the influence of serum citrulline level.Mouse (5 is one group) has been accepted the different ADI of 5.0IU amount and the composition of PEG-ADI.In order to measure the citrulline serum level, mouse is passed through eye socket blood vessel bloodletting (100 μ l).Add isopyknic 50% (w/v) trichoroacetic acid(TCA) after the collection immediately.Centrifugal (13,000xg, 30 minutes) remove precipitation, get supernatant in-70 ℃ of preservations.With full-automatic amino acidanalyser of BeckmanInstruments and reagent, according to the program analysis sample that manufacturers provided.This method is about 2-6 μ M to the sensitivity limit of citrulline, and the repeatability of measured value is in about 8%.Determine the amount of citrulline, the area guestimate below the curve also is expressed as μ mol days.
In Fig. 4, empty circles represents that solid circles is represented ADI-SC-PEG by the amount of the citrulline of natural A DI generation, and hollow square is represented ADI-SS-PEG, and hollow triangle is represented ADI-SPA-PEG, and black triangle is represented side chain PEG-NHS-PEG2.Result shown in Figure 4 shows the effect of the molecular weight decision PEG-ADI composition of PEG.Must use the biocompatibility linking group except that using in vivo, the effect of PEG-ADI composition must not depend on PEG and ADI bonded method or mode.
The result of Fig. 4 shows that also the optimum weight of PEG is 20,000.As if although PEG30 aspect drug effect, 000 is better than PEG20, and 000, PEG30, it is difficult to operation 000 solubility official post.Output depends on enzymic activity and reclaims, for PEG5, and 000 and PEG12,000 is about 90%; To PEG20,000 is about 85%, and to PEG30,000 is about 40%.Therefore, determined as citrulline output, PEG20, the 000th, between output and circulating half-life, reach the equilibrated optimal selection.
The 3rd experiment is used for determining ADI-SS-PEG5,000 and ADI-SS-PEG20, and the 000 pair of serum arginine consumes and the dose response of citrulline output.To the ADI-SS-PEG5 of mouse (5 is one group) single injection 0.05IU, 0.5IU or 5.0IU, 000 or ADI-SS-PEG20,000.As mentioned above shown in time collect serum, carry out amino acid analysis to quantize serum arginine (Fig. 5 A and 5C) and serum citrulline (Fig. 5 B and 5D).The two kinds of all dependent serum arginine minimizing of inductive dose and increases of serum citrulline of formulation.Yet, ADI-SS-PEG20,000 induce effect than ADI-SS-PEG5,000 effect is more obvious, the time length is longer.
The Cytotoxic selectivity of embodiment 4:ADI mediation
Cytotoxic selectivity with some tumour proof arginine deiminase mediations.Particularly, in vitro detection people tumour to ADI-SS-PEG5, the sensitivity of 000 (50ng/ml).Determine the viability of culture after 7 days.Absorb under the situation of trypan blue dyestuff at cell, described culture can be defined as " inhibition " more than 95%.Detected many normal cells yet, comprised endotheliocyte, smooth muscle cell, epithelial cell and inoblast, these cells all are not subjected to ADI-SS-PEG5,000 inhibition.Although arginine deiminase does not have measurable toxicity to normal cell and most of tumour cell, ADI-SS-PEG5,000 has but suppressed whole human melanoma and hepatomas greatly, and these oncocytes are to be purchased from ATCC, MSKCC and Europe.
Table 1: the Cytotoxic specificity of arginine deiminase
Tumor type Tested number of tumors Tumor control rate (%)
Brain ????16 ??0
Colon ????34 ??0
Bladder ????3 ??0
Breast ????12 ??0
Kidney ????5 ??0
Sarcoma ????11 ??64
Hepatoma ????17 ??100
Melanoma ????37 ??100
In one group of parallel laboratory test, separating mRNA from described tumour.Utilize people's argininosuccinic acid synthase cDNA probe to carry out the Northern engram analysis, show the sensitivity of handling at arginine deiminase and can not express between the argininosuccinic acid synthase to have crash consistency.These data show that ADI toxicity can not induce the argininosuccinic acid synthase and cause owing to it.Therefore, these cells can not synthesize arginine from citrulline, can not the necessary protein of synthetically grown.
Embodiment 5: circulating half-life
Shown in Fig. 2 A and 2B, the natural arginine of Balb C mouse (5 is a group) intravenous injection single dose 5.0IU take off imines enzyme or different dosage form through polyethyleneglycol modified arginine deiminase.In order to measure the serum level of arginine and citrulline, mouse is passed through eye socket blood vessel bloodletting (100 μ l).Add isopyknic 50% (w/v) trichoroacetic acid(TCA) after the collection immediately.Centrifugal (13,000xg, 30 minutes) remove precipitation, get supernatant in-70 ℃ of preservations.With full-automatic amino acidanalyser of BeckmanInstruments and reagent, according to the program analysis sample that manufacturers provided.This method is about 6pM to arginic sensitivity limit, and the repeatability of measured value is in about 8%.
After single dose was used natural A DI (solid circles) or ADI-SS-PEG (hollow triangle), the serum arginine-level that detects dose-dependently reduced, shown in the solid circles of Fig. 2 A and the serum citrulline raise, shown in the hollow triangle among Fig. 2 B.Yet the serum arginine reduces and serum raises all is of short duration, promptly recovers normal soon.The transformation period of arginine consumption is summarized in following table.
Table 2: the transformation period that the serum arginine consumes
Compound The transformation period fate
Natural A DI ????1
????ADI-SS-PEG5,000 ????5
????ADI-SS-PEG12,000 ????15
????ADI-SS-PEG20,000 ????20
????ADI-SS-PEG30,000 ????22
This experiment shows that normal cell and tissue can transform back into arginine with citrulline in cell, and melanoma and hepatoma can not, this is because their lack argininosuccinate synthetase.
The antigenicity of the ADI that embodiment 6:PEG modifies
In order to determine natural A DI, ADI-SS-PEG5,000 and ADI SS-PEG20,000 antigenicity is used the method described in the following document, referring to Park, Ahticancer Res., the same and Kamisaki, J.Pharmacol.Exp.Ther., the same.In brief, weekly to natural A DI, the ADI-SS-PEG5 of Balb C mouse (5 is a group) the about 0.5IU of intravenous injection (100 μ g protein), 000 or ADI-SS-PEG20,000, totally 12 weeks.Experiment beginning and the 4th, 8 and 12 weeks with the blood vessel bloodletting (0.05ml) behind the eye socket of described animal.Separation of serum also is stored in-70 ℃.Measure tiring of anti--ADI IgG with ELISA.Add 50 μ g ADI in every hole of 96 hole titer plate, incubation is 4 hours under the room temperature.Wash plate with PBS, then with bovine serum albumin (1mg/ml) bag by with sealing nonspecific proteins binding site, preserve down at 4 ℃ and to spend the night.Dilute mice serum next day, and be added in the hole.After 1 hour, wash plate with PBS, and with coupling the anti-mouse IgG of rabbit of peroxidase be added in the hole.Plate incubation 30 minutes uses the titer plate readout instrument to measure the UV absorbancy of gained then.Potency unit is defined as the high dilution of serum that causes exceeding twice than background absorbancy (about 0.50OD).
The result as shown in Figure 6.The empty circles representative is by the data of the animal gained of the very strong natural A DI of injections of antigens.Solid circles representative has been by having injected ADI-SS-PEG5,000 the data that animal obtained, and the hollow triangle representative is by having injected ADI-SS-PEG20,000 the data that animal obtained.As can be seen from Figure 6, ADI-SS-PEG5,000 and ADI-SS-PEG20,000 antigenicity is markedly inferior to natural A DI.For example, inject natural A DI to 4 times less and cause about 10 6Tire, all do not produce any measurable antibody and inject any PEG-ADI formulation for 4 times.Yet,, produced about 10 after 000 at 8 injection ADI-PEG5 2Tire, and 12 times the injection before, ADI-PEG20,000 does not all induce so immune system response.This result shows that PEG has been attached to ADI this proteic immunne response that gone up passivation.
Embodiment 7: the tumor suppression of human melanoma
In nude mice, measured the influence of PEG-ADI to the human melanoma growth.Give nude mice (5 is a group) injection 10 6SK-mel 2 human melanoma cells, and make described cell grow into the about 3-5 millimeter of diameter of tumor.Mouse or not treated (empty circles), or with the ADI-SS-PEG5 of 5.0IU, 000 (black triangle), ADI-SS-PEG12,000 (hollow triangle) or ADI-SS-PEG20,000 (solid circles) handled once weekly, totally 8 weeks.Measure the size of tumour weekly, the mean diameter of described tumour is shown among Fig. 7.
Fig. 8 represents ADI-SS-PEG20, the effect of 000 pair of three-type-person's melanoma of growing in nude mouse (SK-mel 2, SK-mel 28, M24-met).Give nude mice (5 is a group) injection 10 6SK mel 2, SK mel 28 or M24-met human melanoma cell.Make described tumor growth be about the 3-5 millimeter to diameter.Thereafter, above-mentioned animal is injected the ADI-SS-PEG20 of a 5.0IU, 000 weekly.Result as shown in Figure 8 shows that PEG-ADI has suppressed growth of tumor, and tumour is slowly diminished until completely dissolve.Because tumour does not have argininosuccinate synthetase, they can not synthetic protein (because ADI has removed arginine and tumour can not be synthesized arginine), so this cell " hungry causing death ".
Because the M24-met human melanoma is highly metastatic, inject M24-met human melanoma cell's animal and handled the back execution of 4 weeks, measure the number of metastasis in the animal lung.Control animal on average has 32 metastasis, and uses ADI-SS-PEG20, and the 000 animal kind of handling is without any metastasis.As if this result show, ADI-SS-PEG20, and 000 not only suppresses the transfer of melanomatous growth of primary but also inhibition tumour.
What is interesting is that in the animal subject more than 200, the mean transferred kitchen range number of control group is 49 ± 18, and only has 1 discovery that single metastasis is arranged in treated animal.
Embodiment 8: the inhibition of human liver cell knurl
Detect PEG-ADI and suppressed the ability that the human liver cell knurl is grown in vivo.Give nude mice (5 is a group) injection 10 6Human liver cell knurl SKHepl cell.Make described 2 weeks of tumor growth, use the SS-PEG5 of 5.0IU then, 000-ADI (solid circles), SS-PEG12,000-ADI (black triangle) or SS-PEG20,000-ADI (hollow triangle) handles described animal, and is weekly.The result as shown in Figure 9.Undressed animal (empty circles) is all dead in 3 weeks.On the contrary, as can be seen from Figure 9, the animal of handling with ADI can have the long life-span.The mouse that is survived was sentenced euthanasia after 6 months, postmortem shows that they all do not have tumour.
Surprisingly, it is the most effective that PEG5,000-ADI suppress the hepatoma growth in vivo.The appropriate mechanism of this phenomenon is still unknown.As if work of the present invention is not bound by any theory, uses SS-PEG5, and the albumen of 000-ADI preparation is isolated in liver.The PEG of larger molecular weight is not so, and this may be because the uniqueness of liver endothelium and the PEG-ADI binding substances that the spatial size makes larger molecular weight have been left out.
Embodiment 9: to people's application
With PEG5,000-ADI and PEG20,000-ADI determines its influence to arginine concentration at external and normal human serum incubation together by amino acid analysis, find that wherein this enzyme is active fully, and can with the degraded of kinetics identical in mouse experiment all can detected arginine.0.1ml reaction volume under 37 ℃, carried out 1 hour.
In addition, the arginine in human serum is identical with situation about finding in mouse with the citrulline level.PEG-protein circulates in the people than in mouse the longer time.For example, after adenosine took off imines enzyme, asparaginase, glucocerbrocidase, uriKoxidase, hyperglobulinemia (hemoglobulin) and superoxide dismutase and PEG combines, its circulating half-life was all than long 5 to 10 times of the circulating half-life of same preparation in mouse.This means that the dosage that is used for the people normally is used for 1/5 to 1/10 of mouse dosage.Therefore, PEG-ADI should be than the longer time of circulation in mouse in the people.
Each piece patent, patent application and the publication quoted from this specification sheets all are incorporated herein by reference in full with it.
Except that described herein, described in view of preamble, any change that the present invention made is apparent to those skilled in the art.These modifications fall within the scope of the application's claims too.
Sequence table
＜110〉Phoenix Pharmacologics Inc. (Phoenix Pharmaceologics, Inc.)
＜120〉arginine deiminase of Xiu Shiing (Modified Arginine Deiminase)
<130>?SCT031404-47
<150>?PCT/US01/29184
<151>?2001-09-19
<150>?09/723,546
<151>?2000-11-28
<160>?14
<170>?PatentIn?version?3.2
<210>?1
<211>?410
<212>?PRT
＜213〉mycoplasma arginini (Mycoplasma arginini)
<400>?1
Met?Ser?Val?Phe?Asp?Ser?Lys?Phe?Lys?Gly?Ile?His?Val?Tyr?Ser?Glu
1???????????????5???????????????????10??????????????????15
Ile?Gly?Glu?Leu?Glu?Ser?Val?Leu?Val?His?Glu?Pro?Gly?Arg?Glu?Ile
20??????????????????25??????????????????30
Asp?Tyr?Ile?Thr?Pro?Ala?Arg?Leu?Asp?Glu?Leu?Leu?Phe?Ser?Ala?Ile
35??????????????????40??????????????????45
Leu?Glu?Ser?His?Asp?Ala?Arg?Lys?Glu?His?Lys?Gln?Phe?Val?Ala?Glu
50??????????????????55??????????????????60
Leu?Lys?Ala?Asn?Asp?Ile?Asn?Val?Val?Glu?Leu?Ile?Asp?Leu?Val?Ala
65??????????????????70??????????????????75??????????????????80
Glu?Thr?Tyr?Asp?Leu?Ala?Ser?Gln?Glu?Ala?Lys?Asp?Lys?Leu?Ile?Glu
85??????????????????90??????????????????95
Glu?Phe?Leu?Glu?Asp?Ser?Glu?Pro?Val?Leu?Ser?Glu?Glu?His?Lys?Val
100?????????????????105?????????????????110
Val?Val?Arg?Asn?Phe?Leu?Lys?Ala?Lys?Lys?Thr?Ser?Arg?Lys?Leu?Val
115?????????????????120?????????????????125
Glu?Ile?Met?Met?Ala?Gly?Ile?Thr?Lys?Tyr?Asp?Leu?Gly?Ile?Glu?Ala
130?????????????????135?????????????????140
Asp?His?Glu?Leu?Ile?Val?Asp?Pro?Met?Pro?Asn?Leu?Tyr?Phe?Thr?Arg
145?????????????????150?????????????????155?????????????????160
Asp?Pro?Phe?Ala?Ser?Val?Gly?Asn?Gly?Val?Thr?Ile?His?Tyr?Met?Arg
165?????????????????170?????????????????175
Tyr?Lys?Val?Arg?Gln?Arg?Glu?Thr?Leu?Phe?Ser?Arg?Phe?Val?Phe?Ser
180?????????????????185?????????????????190
Asn?His?Pro?Lys?Leu?Ile?Asn?Thr?Pro?Trp?Tyr?Tyr?Asp?Pro?Ser?Leu
195?????????????????200?????????????????205
Lys?Leu?Ser?Ile?Glu?Gly?Gly?Asp?Val?Phe?Ile?Tyr?Asn?Asn?Asp?Thr
210?????????????????215?????????????????220
Leu?Val?Val?Gly?Val?Ser?Glu?Arg?Thr?Asp?Leu?Gln?Thr?Val?Thr?Leu
225?????????????????230?????????????????235?????????????????240
Leu?Ala?Lys?Asn?Ile?Val?Ala?Asn?Lys?Glu?Cys?Glu?Phe?Lys?Arg?Ile
245?????????????????250?????????????????255
Val?Ala?Ile?Asn?Val?Pro?Lys?Trp?Thr?Asn?Leu?Met?His?Leu?Asp?Thr
260?????????????????265?????????????????270
Trp?Leu?Thr?Met?Leu?Asp?Lys?Asp?Lys?Phe?Leu?Tyr?Ser?Pro?Ile?Ala
275?????????????????280?????????????????285
Asn?Asp?Val?Phe?Lys?Phe?Trp?Asp?Tyr?Asp?Leu?Val?Asn?Gly?Gly?Ala
290?????????????????295?????????????????300
Glu?Pro?Gln?Pro?Val?Glu?Asn?Gly?Leu?Pro?Leu?Glu?Gly?Leu?Leu?Gln
305?????????????????310?????????????????315?????????????????320
Ser?Ile?Ile?Asn?Lys?Lys?Pro?Val?Leu?Ile?Pro?Ile?Ala?Gly?Glu?Gly
325?????????????????330?????????????????335
Ala?Ser?Gln?Met?Glu?Ile?Glu?Arg?Glu?Thr?His?Phe?Asp?Gly?Thr?Asn
340?????????????????345?????????????????350
Tyr?Leu?Ala?Ile?Arg?Pro?Gly?Val?Val?Ile?Gly?Tyr?Ser?Arg?Asn?Glu
355?????????????????360?????????????????365
Lys?Thr?Asn?Ala?Ala?Leu?Glu?Ala?Ala?Gly?Ile?Lys?Val?Leu?Pro?Phe
370?????????????????375?????????????????380
His?Gly?Asn?Gln?Leu?Ser?Leu?Gly?Met?Gly?Asn?Ala?Arg?Cys?Met?Ser
385?????????????????390?????????????????395?????????????????400
Met?Pro?Leu?Ser?Arg?Lys?Asp?Val?Lys?Trp
405?????????????????410
<210>2
<211>410
<212>PRT
＜213〉mycoplasma arthritidis (Mycoplasma arthritidis)
<400>2
Met?Ser?Val?Phe?Asp?Ser?Lys?Phe?Lys?Gly?Ile?His?Val?Tyr?Ser?Glu
1???????????????5???????????????????10??????????????????15
Ile?Gly?Glu?Leu?Glu?Ser?Val?Leu?Val?His?Glu?Pro?Gly?Arg?Glu?Ile
20??????????????????25??????????????????30
Asp?Tyr?Ile?Thr?Pro?Ala?Arg?Leu?Asp?Glu?Leu?Leu?Phe?Ser?Ala?Ile
35??????????????????40??????????????????45
Leu?Glu?Ser?His?Asp?Ala?Arg?Lys?Glu?Gln?Ser?Gln?Phe?Val?Ala?Ile
50??????????????????55??????????????????60
Leu?Lys?Ala?Asn?Asp?Ile?Asn?Val?Val?Glu?Thr?Ile?Asp?Leu?Val?Ala
65??????????????????70??????????????????75??????????????????80
Glu?Thr?Tyr?Asp?Leu?Ala?Ser?Gln?Glu?Ala?Lys?Asp?Arg?Leu?Ile?Glu
85??????????????????90??????????????????95
Glu?Phe?Leu?Glu?Asp?Ser?Glu?Pro?Val?Leu?Ser?Glu?Ala?His?Lys?Lys
100?????????????????105?????????????????110
Val?Val?Arg?Asn?Phe?Leu?Lys?Ala?Lys?Lys?Thr?Ser?Arg?Lys?Leu?Val
115?????????????????120?????????????????125
Glu?Leu?Met?Met?Ala?Gly?Ile?Thr?Lys?Tyr?Asp?Leu?Gly?Val?Glu?Ala
130?????????????????135?????????????????140
Asp?His?Glu?Leu?Ile?Val?Asp?Pro?Met?Pro?Asn?Leu?Tyr?Phe?Thr?Arg
145?????????????????150?????????????????155?????????????????160
Asp?Pro?Phe?Ala?Ser?Val?Gly?Asn?Gly?Val?Thr?Ile?His?Phe?Met?Arg
165?????????????????170?????????????????175
Tyr?Lys?Val?Arg?Arg?Arg?Glu?Thr?Leu?Phe?Ser?Arg?Phe?Val?Phe?Arg
180?????????????????185?????????????????190
Asn?His?Pro?Lys?Leu?Val?Asn?Thr?Pro?Trp?Tyr?Tyr?Asp?Pro?Ala?Met
195?????????????????200?????????????????205
Lys?Leu?Ser?Ile?Glu?Gly?Gly?Asp?Val?Phe?Ile?Tyr?Asn?Asn?Asp?Thr
210?????????????????215?????????????????220
Leu?Val?Val?Gly?Val?Ser?Glu?Arg?Thr?Asp?Leu?Asp?Thr?Val?Thr?Leu
225?????????????????230?????????????????235?????????????????240
Leu?Ala?Lys?Asn?Leu?Val?Ala?Asn?Lys?Glu?Cys?Glu?Phe?Lys?Arg?Ile
245?????????????????250?????????????????255
Val?Ala?Ile?Asn?Val?Pro?Lys?Trp?Thr?Asn?Leu?Met?His?Leu?Asp?Thr
260?????????????????265?????????????????270
Trp?Leu?Thr?Met?Leu?Asp?Lys?Asn?Lys?Phe?Leu?Tyr?Ser?Pro?Ile?Ala
275?????????????????280?????????????????285
Asn?Asp?Val?Phe?Lys?Phe?Trp?Asp?Tyr?Asp?Leu?Val?Asn?Gly?Gly?Ala
290?????????????????295?????????????????300
Glu?Pro?Gln?Pro?Val?Glu?Asn?Gly?Leu?Pro?Leu?Glu?Lys?Leu?Leu?Gln
305?????????????????310?????????????????315?????????????????320
Ser?Ile?Ile?Asn?Lys?Lys?Pro?Val?Leu?Ile?Pro?Ile?Ala?Gly?Glu?Gly
325?????????????????330?????????????????335
Ala?Ser?Gln?Met?Glu?Ile?Glu?Arg?Glu?Thr?His?Phe?Asp?Gly?Thr?Asn
340?????????????????345?????????????????350
Tyr?Ile?Ala?Ile?Arg?Pro?Gly?Val?Val?Ile?Gly?Tyr?Ser?Arg?Asn?Glu
355?????????????????360?????????????????365
Lys?Thr?Asn?Ala?Ala?Leu?Lys?Ala?Ala?Gly?Ile?Lys?Val?Leu?Pro?Phe
370?????????????????375?????????????????380
His?Gly?Asn?Gln?Leu?Ser?Leu?Gly?Met?Gly?Asn?Ala?Arg?Cys?Met?Ser
385?????????????????390?????????????????395?????????????????400
Met?Pro?Leu?Ser?Arg?Lys?Asp?Val?Lys?Trp
405?????????????????410
<210>3
<211>409
<212>PRT
＜213〉mycoplasma hominis (Mycoplasma hominis)
<400>3
Met?Ser?Val?Phe?Asp?Ser?Lys?Phe?Asn?Gly?Ile?His?Val?Tyr?Ser?Glu
1???????????????5???????????????????10??????????????????15
Ile?Gly?Glu?Leu?Glu?Thr?Val?Leu?Val?His?Glu?Pro?Gly?Arg?Glu?Ile
20??????????????????25??????????????????30
Asp?Tyr?Ile?Thr?Pro?Ala?Arg?Leu?Asp?Glu?Leu?Leu?Phe?Ser?Ala?Ile
35??????????????????40??????????????????45
Leu?Glu?Ser?His?Asp?Ala?Arg?Lys?Glu?His?Gln?Ser?Phe?Val?Lys?Ile
50??????????????????55??????????????????60
Met?Lys?Asp?Arg?Gly?Ile?Asn?Val?Val?Glu?Leu?Thr?Asp?Leu?Val?Ala
65??????????????????70??????????????????75??????????????????80
Glu?Thr?Tyr?Asp?Leu?Ala?Ser?Lys?Ala?Ala?Lys?Glu?Glu?Phe?Ile?Glu
85??????????????????90??????????????????95
Thr?Phe?Leu?Glu?Glu?Thr?Val?Pro?Val?Leu?Thr?Glu?Ala?Asn?Lys?Lys
100?????????????????105?????????????????110
Ala?Val?Arg?Ala?Phe?Leu?Leu?Ser?Lys?Pro?Thr?His?Glu?Met?Val?Glu
115?????????????????120?????????????????125
Phe?Met?Met?Ser?Gly?Ile?Thr?Lys?Tyr?Glu?Leu?Gly?Val?Glu?Ser?Glu
130?????????????????135?????????????????140
Asn?Glu?Leu?Ile?Val?Asp?Pro?Met?Pro?Asn?Leu?Tyr?Phe?Thr?Arg?Asp
145?????????????????150?????????????????155?????????????????160
Pro?Phe?Ala?Ser?Val?Gly?Asn?Gly?Val?Thr?Ile?His?Phe?Met?Arg?Tyr
165?????????????????170?????????????????175
Ile?Val?Arg?Arg?Arg?Glu?Thr?Leu?Phe?Ala?Arg?Phe?Val?Phe?Arg?Asn
180?????????????????185?????????????????190
His?Pro?Lys?Leu?Val?Lys?Thr?Pro?Trp?Tyr?Tyr?Asp?Pro?Ala?Met?Lys
195?????????????????200?????????????????205
Met?Pro?Ile?Glu?Gly?Gly?Asp?Val?Phe?Ile?Tyr?Asn?Asn?Glu?Thr?Leu
210?????????????????215?????????????????220
Val?Val?Gly?Val?Ser?Glu?Arg?Thr?Asp?Leu?Asp?Thr?Ile?Thr?Leu?Leu
225?????????????????230?????????????????235?????????????????240
Ala?Lys?Asn?Ile?Lys?Ala?Asn?Lys?Glu?Val?Glu?Phe?Lys?Arg?Ile?Val
245?????????????????250?????????????????255
Ala?Ile?Asn?Val?Pro?Lys?Trp?Thr?Asn?Leu?Met?His?Leu?Asp?Thr?Trp
260?????????????????265?????????????????270
Leu?Thr?Met?Leu?Asp?Lys?Asn?Lys?Phe?Leu?Tyr?Ser?Pro?Ile?Ala?Asn
275?????????????????280?????????????????285
Asp?Val?Phe?Lys?Phe?Trp?Asp?Tyr?Asp?Leu?Val?Asn?Gly?Gly?Ala?Glu
290?????????????????295?????????????????300
Pro?Gln?Pro?Gln?Leu?Asn?Gly?Leu?Pro?Leu?Asp?Lys?Leu?Leu?Ala?Ser
305?????????????????310?????????????????315?????????????????320
Ile?Ile?Asn?Lys?Glu?Pro?Val?Leu?Ile?Pro?Ile?Gly?Gly?Ala?Gly?Ala
325?????????????????330?????????????????335
Thr?Glu?Met?Glu?Ile?Ala?Arg?Glu?Thr?Asn?Phe?Asp?Gly?Thr?Asn?Tyr
340?????????????????345?????????????????350
Leu?Ala?Ile?Lys?Pro?Gly?Leu?Val?Ile?Gly?Tyr?Asp?Arg?Asn?Glu?Lys
355?????????????????360?????????????????365
Thr?Asn?Ala?Ala?Leu?Lys?Ala?Ala?Gly?Ile?Thr?Val?Leu?Pro?Phe?His
370?????????????????375?????????????????380
Gly?Asn?Gln?Leu?Ser?Leu?Gly?Met?Gly?Asn?Ala?Arg?Cys?Met?Ser?Met
385?????????????????390?????????????????395?????????????????400
Pro?Leu?Ser?Arg?Lys?Asp?Val?Lys?Trp
405
<210>4
<211>25
<212>DNA
＜213〉mycoplasma arginini (Mycoplasma arginini)
<400>4
gggatccatg?tctgtatttg?acagt??????????????????????????????????????????25
<210>5
<211>33
<212>DNA
＜213〉mycoplasma arginini (Mycoplasma arginini)
<400>5
tgaggatcct?tactaccact?taacatcttt?acg?????????????????????????????????33
<210>6
<211>411
<212>PRT
＜213〉streptococcus pyogenes (Steptococcus pyogenes)
<400>6
Met?Thr?Ala?Gln?Thr?Pro?Ile?His?Val?Tyr?Ser?Glu?Ile?Gly?Lys?Leu
1???????????????5???????????????????10??????????????????15
Lys?Lys?Val?Leu?Leu?His?Arg?Pro?Gly?Lys?Glu?Ile?Glu?Asn?Leu?Met
20??????????????????25??????????????????30
Pro?Asp?Tyr?Leu?Glu?Arg?Leu?Leu?Phe?Asp?Asp?Ile?Pro?Phe?Leu?Glu
35??????????????????40??????????????????45
Asp?Ala?Gln?Lys?Glu?His?Asp?Ala?Phe?Ala?Gln?Ala?Leu?Arg?Asp?Glu
50??????????????????55??????????????????60
Gly?Ile?Glu?Val?Leu?Tyr?Leu?Glu?Thr?Leu?Ala?Ala?Glu?Ser?Leu?Val
65??????????????????70??????????????????75??????????????????80
Thr?Pro?Glu?Ile?Arg?Glu?Ala?Phe?Ile?Asp?Glu?Tyr?Leu?Ser?Glu?Ala
85??????????????????90??????????????????95
Asn?Ile?Arg?Gly?Arg?Ala?Thr?Lys?Lys?Ala?Ile?Arg?Glu?Leu?Leu?Met
100?????????????????105?????????????????110
Ala?Ile?Glu?Asp?Asn?Gln?Glu?Leu?Ile?Glu?Lys?Thr?Met?Ala?Gly?Val
115?????????????????120?????????????????125
Gln?Lys?Ser?Glu?Leu?Pro?Glu?Ile?Pro?Ala?Ser?Glu?Lys?Gly?Leu?Thr
130?????????????????135?????????????????140
Asp?Leu?Val?Glu?Ser?Asn?Tyr?Pro?Phe?Ala?Ile?Asp?Pro?Met?Pro?Asn
145?????????????????150?????????????????155?????????????????160
Leu?Tyr?Phe?Thr?Arg?Asp?Pro?Phe?Ala?Thr?Ile?Gly?Thr?Gly?Val?Ser
165?????????????????170?????????????????175
Leu?Asn?His?Met?Phe?Ser?Glu?Thr?Arg?Asn?Arg?Glu?Thr?Leu?Tyr?Gly
180?????????????????185?????????????????190
Lys?Tyr?Ile?Phe?Thr?His?His?Pro?Ile?Tyr?Gly?Gly?Gly?Lys?Val?Pro
195?????????????????200?????????????????205
Met?Val?Tyr?Asp?Arg?Asn?Glu?Thr?Thr?Arg?Ile?Glu?Gly?Gly?Asp?Glu
210?????????????????215?????????????????220
Leu?Val?Leu?Ser?Lys?Asp?Val?Leu?Ala?Val?Gly?Ile?Ser?Gln?Arg?Thr
225?????????????????230?????????????????235?????????????????240
Asp?Ala?Ala?Ser?Ile?Glu?Lys?Leu?Leu?Val?Asn?Ile?Phe?Lys?Gln?Asn
245?????????????????250?????????????????255
Leu?Gly?Phe?Lys?Lys?Val?Leu?Ala?Phe?Glu?Phe?Ala?Asn?Asn?Arg?Lys
260?????????????????265?????????????????270
Phe?Met?His?Leu?Asp?Thr?Val?Phe?Thr?Met?Val?Asp?Tyr?Asp?Lys?Phe
275?????????????????280?????????????????285
Thr?Ile?His?Pro?Glu?Ile?Glu?Gly?Asp?Leu?Arg?Val?Tyr?Ser?Val?Thr
290?????????????????295?????????????????300
Tyr?Asp?Asn?Glu?Glu?Leu?His?Ile?Val?Glu?Glu?Lys?Gly?Asp?Leu?Ala
305?????????????????310?????????????????315?????????????????320
Glu?Leu?Leu?Ala?Ala?Asn?Leu?Gly?Val?Glu?Lys?Val?Asp?Leu?Ile?Arg
325?????????????????330?????????????????335
Cys?Gly?Gly?Asp?Asn?Leu?Val?Ala?Ala?Gly?Arg?Glu?Gln?Trp?Asn?Asp
340?????????????????345?????????????????350
Gly?Ser?Asn?Thr?Leu?Thr?Ile?Ala?Pro?Gly?Val?Val?Val?Val?Tyr?Ash
355?????????????????360?????????????????365
Arg?Asn?Thr?Ile?Thr?Asn?Ala?Ile?Leu?Glu?Ser?Lys?Gly?Leu?Lys?Leu
370?????????????????375?????????????????380
Ile?Lys?Ile?His?Gly?Ser?Glu?Leu?Val?Arg?Gly?Arg?Gly?Gly?Pro?Arg
385?????????????????390?????????????????395?????????????????400
Cys?Met?Ser?Met?Pro?Phe?Glu?Arg?Glu?Asp?Ile
405?????????????????410
<210>7
<211>409
<212>PRT
＜213〉streptococcus pneumoniae (Steptococcus pneumoniae)
<400>7
Met?Ser?Ser?His?Pro?Ile?Gln?Val?Phe?Ser?Glu?Ile?Gly?Lys?Leu?Lys
1???????????????5???????????????????10??????????????????15
Lys?Val?Met?Leu?His?Arg?Pro?Gly?Lys?Glu?Leu?Glu?Asn?Leu?Leu?Pro
20??????????????????25??????????????????30
Asp?Tyr?Leu?Glu?Arg?Leu?Leu?Phe?Asp?Asp?Ile?Pro?Phe?Leu?Glu?Asp
35??????????????????40??????????????????45
Ala?Gln?Lys?Glu?His?Asp?Ala?Phe?Ala?Gln?Ala?Leu?Arg?Asp?Glu?Gly
50??????????????????55??????????????????60
Ile?Glu?Val?Leu?Tyr?Leu?Glu?Gln?Leu?Ala?Ala?Glu?Ser?Leu?Thr?Ser
65??????????????????70??????????????????75??????????????????80
Pro?Glu?Ile?Arg?Asp?Gln?Phe?Ile?Glu?Glu?Tyr?Leu?Asp?Glu?Ala?Asn
85??????????????????90??????????????????95
Ile?Arg?Asp?Arg?Gln?Thr?Lys?Val?Ala?Ile?Arg?Glu?Leu?Leu?His?Gly
100?????????????????105?????????????????110
Ile?Lys?Asp?Asn?Gln?Glu?Leu?Val?Glu?Lys?Thr?Met?Ala?Gly?Ile?Gln
115?????????????????120?????????????????125
Lys?Val?Glu?Leu?Pro?Glu?IIe?Pro?Asp?Glu?Ala?Lys?Asp?Leu?Thr?Asp
130?????????????????135?????????????????140
Leu?Val?Glu?Ser?Glu?Tyr?Pro?Phe?Ala?Ile?Asp?Pro?Met?Pro?Asn?Leu
145?????????????????150?????????????????155?????????????????160
Tyr?Phe?Thr?Arg?Asp?Pro?Phe?Ala?Thr?Ile?Gly?Asn?Ala?Val?Ser?Leu
165?????????????????170?????????????????175
Asn?His?Met?Phe?Ala?Asp?Thr?Arg?Asn?Arg?Glu?Thr?Leu?Tyr?Gly?Lys
180?????????????????185?????????????????190
Tyr?Ile?Phe?Lys?Tyr?His?Pro?Ile?Tyr?Gly?Gly?Lys?Val?Asp?Leu?Val
195?????????????????200?????????????????205
Tyr?Asn?Arg?Glu?Glu?Asp?Thr?Arg?Ile?Glu?Gly?Gly?Asp?Glu?Leu?Val
210?????????????????215?????????????????220
Leu?Ser?Lys?Asp?Val?Leu?Ala?Val?Gly?Ile?Ser?Gln?Arg?Thr?Asp?Ala
225?????????????????230?????????????????235?????????????????240
Ala?Ser?Ile?Glu?Lys?Leu?Leu?Val?Asn?Ile?Phe?Lys?Lys?Asn?Val?Gly
245?????????????????250?????????????????255
Phe?Lys?Lys?Val?Leu?Ala?Phe?Glu?Phe?Ala?Asn?Asn?Arg?Lys?Phe?Met
260?????????????????265?????????????????270
His?Leu?Asp?Thr?Val?Phe?Thr?Met?Val?Asp?Tyr?Asp?Lys?Phe?Thr?Ile
275?????????????????280?????????????????285
His?Pro?Glu?Ile?Glu?Gly?Asp?Leu?His?Val?Tyr?Ser?Val?Thr?Tyr?Glu
290?????????????????295?????????????????300
Asn?Glu?Lys?Leu?Lys?Ile?Val?Glu?Glu?Lys?Gly?Asp?Leu?Ala?Glu?Leu
305?????????????????310?????????????????315?????????????????320
Leu?Ala?Gln?Asn?Leu?Gly?Val?Glu?Lys?Val?His?Leu?Ile?Arg?Cys?Gly
325?????????????????330?????????????????335
Gly?Gly?Asn?Ile?Val?Ala?Ala?Ala?Arg?Glu?Gln?Trp?Asn?Asp?Gly?Ser
340?????????????????345?????????????????350
Asn?Thr?Leu?Thr?Ile?Ala?Pro?Gly?Val?Val?Val?Val?Tyr?Asp?Arg?Asn
355?????????????????360?????????????????365
Thr?Val?Thr?Asn?Lys?Ile?Leu?Glu?Glu?Tyr?Gly?Leu?Arg?Leu?Ile?Lys
370?????????????????375?????????????????380
Ile?Arg?Gly?Ser?Glu?Leu?Val?Arg?Gly?Arg?Gly?Gly?Pro?Arg?Cys?Met
385?????????????????390?????????????????395?????????????????400
Ser?Met?Pro?Phe?Glu?Arg?Glu?Glu?Val
405
<210>8
<211>410
<212>PRT
＜213〉B. burgdorferi (Borrelia burgdorferi)
<400>8
Met?Glu?Glu?Glu?Tyr?Leu?Asn?Pro?Ile?Asn?Ile?Phe?Ser?Glu?Ile?Gly
1???????????????5???????????????????10??????????????????15
Arg?Leu?Lys?Lys?Val?Leu?Leu?His?Arg?Pro?Gly?Glu?Glu?Leu?Glu?Asn
20??????????????????25??????????????????30
Leu?Thr?Pro?Leu?IIe?Met?Lys?Asn?Phe?Leu?Phe?Asp?Asp?Ile?Pro?Tyr
35??????????????????40??????????????????45
Leu?Lys?Val?Ala?Arg?Gln?Glu?His?Glu?Val?Phe?Val?Asn?Ile?Leu?Lys
50??????????????????55??????????????????60
Asp?Asn?Ser?Val?Glu?Ile?Glu?Tyr?Val?Glu?Asp?Leu?Val?Ser?Glu?Val
65??????????????????70??????????????????75??????????????????80
Leu?Ala?Ser?Ser?Val?Ala?Leu?Lys?Asn?Lys?Phe?Ile?Ser?Gln?Phe?Ile
85??????????????????90??????????????????95
Leu?Glu?Ala?Glu?Ile?Lys?Thr?Asp?Gly?Val?Ile?Asn?Ile?Leu?Lys?Asp
100?????????????????105?????????????????110
Tyr?Phe?Ser?Asn?Leu?Thr?Val?Asp?Asn?Met?Val?Ser?Lys?Met?Ile?Ser
115?????????????????120?????????????????125
Gly?Val?Ala?Arg?Glu?Glu?Leu?Lys?Asp?Cys?Glu?Phe?Ser?Leu?Asp?Asp
130?????????????????135?????????????????140
Trp?Val?Asn?Gly?Ser?Ser?Leu?Phe?Val?Ile?Asp?Pro?Met?Pro?Asn?Val
145?????????????????150?????????????????155?????????????????160
Leu?Phe?Thr?Arg?Asp?Pro?Phe?Ala?Ser?Ile?Gly?Asn?Gly?Ile?Thr?Ile
165?????????????????170?????????????????175
Asn?Lys?Met?Tyr?Thr?Lys?Val?Arg?Arg?Arg?Glu?Thr?Ile?Phe?Ala?Glu
180?????????????????185?????????????????190
Tyr?Ile?Phe?Lys?Tyr?His?Ser?Ala?Tyr?Lys?Glu?Asn?Val?Pro?Ile?Trp
195?????????????????200?????????????????205
Phe?Asn?Arg?Trp?Glu?Glu?Thr?Ser?Leu?Glu?Gly?Gly?Asp?Glu?Phe?Val
210?????????????????215?????????????????220
Leu?Asn?Lys?Asp?Leu?Leu?Val?Ile?Gly?Ile?Ser?Glu?Arg?Thr?Glu?Ala
225?????????????????230?????????????????235?????????????????240
Gly?Ser?Val?Glu?Lys?Leu?Ala?Ala?Ser?Leu?Phe?Lys?Asn?Lys?Ala?Pro
245?????????????????250?????????????????255
Phe?Ser?Thr?Ile?Leu?Ala?Phe?Lys?Ile?Pro?Lys?Asn?Arg?Ala?Tyr?Met
260?????????????????265?????????????????270
His?Leu?Asp?Thr?Val?Phe?Thr?Gln?Ile?Asp?Tyr?Ser?Val?Phe?Thr?Ser
275?????????????????280?????????????????285
Phe?Thr?Ser?Asp?Asp?Met?Tyr?Phe?Ser?Ile?Tyr?Val?Leu?Thr?Tyr?Asn
290?????????????????295?????????????????300
Ser?Asn?Ser?Asn?Lys?Ile?Asn?Ile?Lys?Lys?Glu?Lys?Ala?Lys?Leu?Lys
305?????????????????310?????????????????315?????????????????320
Asp?Val?Leu?Ser?Phe?Tyr?Leu?Gly?Arg?Lys?Ile?Asp?Ile?Ile?Lys?Cys
325?????????????????330?????????????????335
Ala?Gly?Gly?Asp?Leu?Ile?His?Gly?Ala?Arg?Glu?Gln?Trp?Asn?Asp?Gly
340?????????????????345?????????????????350
Ala?Asn?Val?Leu?Ala?Ile?Ala?Pro?Gly?Glu?Val?Ile?Ala?Tyr?Ser?Arg
355?????????????????360?????????????????365
Asn?His?Val?Thr?Asn?Lys?Leu?Phe?Glu?Glu?Asn?Gly?Ile?Lys?Val?His
370?????????????????375?????????????????380
Arg?Ile?Pro?Ser?Ser?Glu?Leu?Ser?Arg?Gly?Arg?Gly?Gly?Pro?Arg?Cys
385?????????????????390?????????????????395?????????????????400
Met?Ser?Met?Ser?Leu?Val?Arg?Glu?Asp?Ile
405?????????????????410
<210>9
<211>409
<212>PRT
＜213〉Ah's borrelia burgdorferi (Borrelia afzelii)
<400>9
Met?Glu?Glu?Tyr?Leu?Asn?Pro?Ile?Asn?Ile?Phe?Ser?Glu?Ile?Gly?Arg
1???????????????5???????????????????10??????????????????15
Leu?Lys?Lys?Val?Leu?Leu?His?Arg?Pro?Gly?Glu?Glu?Leu?Glu?Asn?Leu
20??????????????????25??????????????????30
Thr?Pro?Phe?Ile?Met?Lys?Asn?Phe?Leu?Phe?Asp?Asp?Ile?Pro?Tyr?Leu
35??????????????????40??????????????????45
Glu?Val?Ala?Arg?Gln?Glu?His?Glu?Val?Phe?Ala?Ser?Ile?Leu?Lys?Asn
50??????????????????55??????????????????60
Asn?Leu?Val?Glu?Ile?Glu?Tyr?Ile?Glu?Asp?Leu?Ile?Ser?Glu?Val?Leu
65??????????????????70??????????????????75??????????????????80
Val?Ser?Ser?Val?Ala?Leu?Glu?Asn?Lys?Phe?Ile?Ser?Gln?Phe?Ile?Leu
85??????????????????90??????????????????95
Glu?Ala?Glu?Ile?Lys?Thr?Asp?Phe?Thr?Ile?Asn?Leu?Leu?Lys?Asp?Tyr
100?????????????????105?????????????????110
Phe?Ser?Ser?Leu?Thr?Ile?Asp?Asn?Met?Ile?Ser?Lys?Met?Ile?Ser?Gly
115?????????????????120?????????????????125
Val?Val?Thr?Glu?Glu?Leu?Lys?Asn?Tyr?Thr?Ser?Ser?Leu?Asp?Asp?Leu
130?????????????????135?????????????????140
Val?Asn?Gly?Ala?Asn?Leu?Phe?Ile?Ile?Asp?Pro?Met?Pro?Asn?Val?Leu
145?????????????????150????????????????155??????????????????160
Phe?Thr?Arg?Asp?Pro?Phe?Ala?Ser?Ile?Gly?Asn?Gly?Val?Thr?Ile?Asn
165?????????????????170?????????????????175
Lys?Met?Phe?Thr?Lys?Val?Arg?Gln?Arg?Glu?Thr?Ile?Phe?Ala?Glu?Tyr
180?????????????????185?????????????????190
Ile?Phe?Lys?Tyr?His?Pro?Val?Tyr?Lys?Glu?Asn?Val?Pro?Ile?Trp?Leu
195?????????????????200?????????????????205
Asn?Arg?Trp?Glu?Glu?Ala?Ser?Leu?Glu?Gly?Gly?Asp?Glu?Leu?Val?Leu
210?????????????????215?????????????????220
Ash?Lys?Gly?Leu?Leu?Val?Ile?Gly?Ile?Ser?Glu?Arg?Thr?Glu?Ala?Lys
225?????????????????230?????????????????235?????????????????240
Ser?Val?Glu?Lys?Leu?Ala?Ile?Ser?Leu?Phe?Lys?Asn?Lys?Thr?Ser?Phe
245?????????????????250?????????????????255
Asp?Thr?Ile?Leu?Ala?Phe?Gln?Ile?Pro?Lys?Asn?Arg?Ser?Tyr?Met?His
260?????????????????265?????????????????270
Leu?Asp?Thr?Val?Phe?Thr?Gln?Ile?Asp?Tyr?Ser?Val?Phe?Thr?Ser?Phe
275?????????????????280?????????????????285
Thr?Ser?Asp?Asp?Met?Tyr?Phe?Ser?Ile?Tyr?Val?Leu?Thr?Tyr?Asn?Pro
290?????????????????295?????????????????300
Ser?Ser?Ser?Lys?Ile?His?Ile?Lys?Lys?Glu?Lys?Ala?Arg?Ile?Lys?Asp
305?????????????????310?????????????????315?????????????????320
Val?Leu?Ser?Phe?Tyr?Leu?Gly?Arg?Lys?Ile?Asp?Ile?Ile?Lys?Cys?Ala
325?????????????????330?????????????????335
Gly?Gly?Asp?Leu?Ile?His?Gly?Ala?Arg?Glu?Gln?Trp?Asn?Asp?Gly?Ala
340?????????????????345?????????????????350
Asn?Val?Leu?Ala?Ile?Ala?Pro?Gly?Glu?Ile?Ile?Ala?Tyr?Ser?Arg?Asn
355?????????????????360?????????????????365
His?Val?Thr?Asn?Lys?Leu?Phe?Glu?Glu?Asn?Gly?Ile?Lys?Val?His?Arg
370?????????????????375?????????????????380
Ile?Pro?Ser?Ser?Glu?Leu?Ser?Arg?Gly?Arg?Gly?Gly?Pro?Arg?Cys?Met
385?????????????????390?????????????????395?????????????????400
Ser?Met?Pro?Leu?Ile?Arg?Glu?Asp?Ile
405
<210>10
<211>580
<212>PRT
<213>Qiardia?intestinalis
<400>10
Met?Thr?Asp?Phe?Ser?Lys?Asp?Lys?Glu?Lys?Leu?Ala?Gln?Ala?Thr?Gln
l???????????????5???????????????????10??????????????????15
Gly?Gly?Glu?Asn?Glu?Arg?Ala?Glu?Ile?Val?Val?Val?His?Leu?Pro?Gln
20??????????????????25??????????????????30
Gly?Thr?Ser?Phe?Leu?Thr?Ser?Leu?Asn?Pro?Glu?Gly?Asn?Leu?Leu?Glu
35??????????????????40??????????????????45
Glu?Pro?Ile?Cys?Pro?Asp?Glu?Leu?Arg?Arg?Asp?His?Glu?Gly?Phe?Gln
50??????????????????55??????????????????60
Ala?Val?Leu?Lys?Glu?Lys?Gly?Cys?Arg?Val?Tyr?Met?Pro?Tyr?Asp?Val
65??????????????????70??????????????????75??????????????????80
Leu?Ser?Glu?Ala?Ser?Pro?Ala?Glu?Arg?Glu?Val?Leu?Met?Asp?Gln?Ala
85??????????????????90??????????????????95
Met?Ala?Ser?Leu?Lys?Tyr?Glu?Leu?His?Ala?Thr?Gly?Ala?Arg?Ile?Thr
100?????????????????105?????????????????110
Pro?Lys?Met?Lys?Tyr?Cys?Val?Ser?Asp?Glu?Tyr?Lys?Arg?Lys?Val?Leu
115?????????????????120?????????????????125
Ser?Ala?Leu?Ser?Thr?Arg?Asn?Leu?Val?Asp?Val?Ile?Leu?Ser?Glu?Pro
130?????????????????135?????????????????140
Val?Ile?His?Leu?Ala?Pro?Gly?Val?Arg?Asn?Thr?Ala?Leu?Val?Thr?Asn
145?????????????????150?????????????????155?????????????????160
Ser?Val?Glu?Ile?His?Asp?Ser?Asn?Asn?Met?Val?Phe?Met?Arg?Asp?Gln
165?????????????????170?????????????????175
Gln?Ile?Thr?Thr?Arg?Arg?Gly?Ile?Val?Met?Gly?Gln?Phe?Gln?Ala?Pro
180?????????????????185?????????????????190
Gln?Arg?Arg?Arg?Glu?Gln?Val?Leu?Ala?Leu?Ile?Phe?Trp?Lys?Arg?Leu
195?????????????????200?????????????????205
Gly?Ala?Arg?Val?Val?Gly?Asp?Cys?Arg?Glu?Gly?Gly?Pro?His?Cys?Met
210?????????????????215?????????????????220
Leu?Glu?Gly?Gly?Asp?Phe?Val?Pro?Val?Ser?Pro?Gly?Leu?Ala?Met?Met
225?????????????????230?????????????????235?????????????????240
Gly?Val?Gly?Leu?Arg?Ser?Thr?Tyr?Val?Gly?Ala?Gln?Tyr?Leu?Met?Ser
245?????????????????250?????????????????255
Lys?Asp?Leu?Leu?Gly?Thr?Arg?Arg?Phe?Ala?Val?Val?Lys?Asp?Cys?Phe
260?????????????????265?????????????????270
Asp?Gln?His?Gln?Asp?Arg?Met?His?Leu?Asp?Cys?Thr?Phe?Ser?Val?Leu
275?????????????????280?????????????????285
His?Asp?Lys?Leu?Val?Val?Leu?Asp?Asp?Tyr?Ile?Cys?Ser?Gly?Met?Gly
290?????????????????295??????????????????300
Leu?Arg?Tyr?Val?Asp?Glu?Trp?lle?Asp?Val?Gly?Ala?Asp?Ala?Val?Lys
305?????????????????310?????????????????315?????????????????320
Lys?Ala?Lys?Ser?Ser?Ala?Val?Thr?Cys?Gly?Asn?Tyr?Val?Leu?Ala?Lys
325?????????????????330?????????????????335
Ala?Asn?Val?Glu?Phe?Gln?Gln?Trp?Leu?Ser?Glu?Asn?Gly?Tyr?Thr?Ile
340?????????????????345?????????????????350
Val?Arg?Ile?Pro?His?Glu?Tyr?Gln?Leu?Ala?Tyr?Gly?Cys?Asn?Asn?Leu
355?????????????????360?????????????????365
Asn?Leu?Gly?Asn?Asn?Cys?Val?Leu?Ser?Val?His?Gln?Pro?Thr?Val?Asp
370?????????????????375?????????????????380
Phe?Ile?Lys?Ala?Asp?Pro?Ala?Tyr?Ile?Ser?Tyr?Cys?Lys?Ser?Asn?Asn
385?????????????????390?????????????????395?????????????????400
Leu?Pro?Asn?Gly?Leu?Asp?Leu?Val?Tyr?Val?Pro?Phe?Arg?Gly?Ile?Thr
405?????????????????410?????????????????415
Arg?Met?Tyr?Gly?Ser?Leu?His?Cys?Ala?Ser?Gln?Val?Val?Tyr?Arg?Thr
420?????????????????425?????????????????430
Pro?Leu?Ala?Pro?Ala?Ala?Val?Lys?Ala?Cys?Glu?Gln?Glu?Gly?Asp?Gly
435?????????????????440?????????????????445
Ile?Ala?Ala?Ile?Tyr?Glu?Lys?Asn?Gly?Glu?Pro?Val?Asp?Ala?Ala?Gly
450?????????????????455?????????????????460
Lys?Lys?Phe?Asp?Cys?Val?Ile?Tyr?Ile?Pro?Ser?Ser?Val?Asp?Asp?Leu
465?????????????????470?????????????????475?????????????????480
Ile?Asp?Gly?Leu?Lys?Ile?Asn?Leu?Arg?Asp?Asp?Ala?Ala?Pro?Ser?Arg
485?????????????????490?????????????????495
Glu?Ile?Ile?Ala?Asp?Ala?Tyr?Gly?Leu?Tyr?Gln?Lys?Leu?Val?Ser?Glu
500?????????????????505?????????????????510
Gly?Arg?Val?Pro?Tyr?Ile?Thr?Trp?Arg?Met?Pro?Ser?Met?Pro?Val?Val
515?????????????????520?????????????????525
Ser?Leu?Lys?Gly?Ala?Ala?Lys?Ala?Gly?Ser?Leu?Lys?Ala?Val?Leu?Asp
530?????????????????535?????????????????540
Lys?Ile?Pro?Gln?Leu?Thr?Pro?Phe?Thr?Pro?Lys?Ala?Val?Glu?Gly?Ala
545?????????????????550?????????????????555?????????????????560
Pro?Ala?Ala?Tyr?Thr?Arg?Tyr?Leu?Gly?Leu?Glu?Gln?Ala?Asp?Ile?Cys
565?????????????????570?????????????????575
Val?Asp?Ile?Lys
580
<210>11
<211>413
<212>PRT
＜213〉bacillus aerogenes capsulatus (Clostridium perfringens)
<400>11
Met?Arg?Asp?Asp?Arg?Ala?Leu?Asn?Val?Thr?Ser?Glu?Ile?Gly?Arg?Leu
1???????????????5???????????????????10??????????????????15
Lys?Thr?Val?Leu?Leu?His?Arg?Pro?Gly?Glu?Glu?Ile?Glu?Asn?Leu?Thr
20??????????????????25??????????????????30
Pro?Asp?Leu?Leu?Asp?Arg?Leu?Leu?Phe?Asp?Asp?Ile?Pro?Tyr?Leu?Lys
35??????????????????40??????????????????45
Val?Ala?Arg?Glu?Glu?His?Asp?Ala?Phe?Ala?Gln?Thr?Leu?Arg?Glu?Ala
50??????????????????55??????????????????60
Gly?Val?Glu?Val?Leu?Tyr?Leu?Glu?Val?Leu?Ala?Ala?Glu?Ala?Ile?Glu
65??????????????????70??????????????????75??????????????????80
Thr?Ser?Asp?Glu?Val?Lys?Gln?Gln?Phe?Ile?Ser?Glu?Phe?Ile?Asp?Glu
85??????????????????90??????????????????95
Ala?Gly?Val?Glu?Ser?Glu?Arg?Leu?Lys?Glu?Ala?Leu?Ile?Glu?Tyr?Phe
100?????????????????105?????????????????110
Asn?Ser?Phe?Ser?Asp?Asn?Lys?Ala?Met?Val?Asp?Lys?Met?Met?Ala?Gly
115?????????????????120?????????????????125
Val?Arg?Lys?Glu?Glu?Leu?Lys?Asp?Tyr?His?Arg?Glu?Ser?Leu?Tyr?Asp
130?????????????????135?????????????????140
Gln?Val?Asn?Asn?Val?Tyr?Pro?Phe?Val?Cys?Asp?Pro?Met?Pro?Asn?Leu
145?????????????????150?????????????????155?????????????????160
Tyr?Phe?Thr?Arg?Glu?Pro?Phe?Ala?Thr?Ile?Gly?His?Gly?Ile?Thr?Leu
165?????????????????170?????????????????175
Asn?His?Met?Arg?Thr?Asp?Thr?Arg?Asn?Arg?Glu?Thr?Ile?Phe?Ala?Lys
180?????????????????185?????????????????190
Tyr?Ile?Phe?Arg?His?His?Pro?Arg?Phe?Glu?Gly?Lys?Asp?Ile?Pro?Phe
195?????????????????200?????????????????205
Trp?Phe?Asn?Arg?Asn?Asp?Lys?Thr?Ser?Leu?Glu?Gly?Gly?Asp?Glu?Leu
210?????????????????215?????????????????220
Ile?Leu?Ser?Lys?Glu?Ile?Leu?Ala?Val?Gly?Ile?Ser?Gln?Arg?Thr?Asp
225?????????????????230?????????????????235?????????????????240
Ser?Ala?Ser?Val?Glu?Lys?Leu?Ala?Lys?Lys?Leu?Leu?Tyr?Tyr?Pro?Asp
245?????????????????250?????????????????255
Thr?Ser?Phe?Lys?Thr?Val?Leu?Ala?Phe?Lys?Ile?Pro?Val?Ser?Arg?Ala
260?????????????????265?????????????????270
Phe?Met?His?Leu?Asp?Thr?Val?Phe?Thr?Gln?Val?Asp?Tyr?Asp?Lys?Phe
275?????????????????280?????????????????285
Thr?Val?His?Pro?Gly?Ile?Val?Gly?Pro?Leu?Glu?Val?Tyr?Ala?Leu?Thr
290?????????????????295?????????????????300
Lys?Asp?Pro?Glu?Asn?Asp?Gly?Gln?Leu?Leu?Val?Thr?Glu?Glu?Val?Asp
305?????????????????310?????????????????315?????????????????320
Thr?Leu?Glu?Asn?Ile?Leu?Lys?Lys?Tyr?Leu?Asp?Arg?Asp?Ile?Lys?Leu
325?????????????????330?????????????????335
Ile?Lys?Cys?Gly?Gly?Gly?Asp?Glu?Ile?Ile?Ala?Ala?Arg?Glu?Gln?Trp
340?????????????????345?????????????????350
Asn?Asp?Gly?Ser?Asn?Thr?Leu?Ala?Ile?Ala?Pro?Gly?Glu?Val?Val?Val
355?????????????????360?????????????????365
Tyr?Ser?Arg?Asn?Tyr?Val?Thr?Asn?Glu?Ile?Leu?Glu?Lys?Glu?Gly?Ile
370?????????????????375?????????????????380
Lys?Leu?His?Val?Ile?Pro?Ser?Ser?Glu?Leu?Ser?Arg?Gly?Arg?Gly?Gly
385?????????????????390?????????????????395?????????????????400
Pro?Arg?Cys?Met?Ser?Met?Pro?Leu?Ile?Arg?Glu?Asp?Leu
405?????????????????410
<210>12
<211>413
<212>PRT
＜213〉Bacillus licheniformis (Bacillus licheniformis)
<400>12
Met?Ile?Met?Thr?Thr?Pro?Ile?His?Val?Tyr?Ser?Glu?Ile?Gly?Pro?Leu
1???????????????5???????????????????10??????????????????15
Lys?Thr?Val?Met?Leu?Lys?Arg?Pro?Gly?Arg?Glu?Leu?Glu?Asn?Leu?Thr
20??????????????????25??????????????????30
Pro?Glu?Tyr?Leu?Glu?Arg?Leu?Leu?Phe?Asp?Asp?Ile?Pro?Phe?Leu?Pro
35??????????????????40??????????????????45
Ala?Val?Gln?Lys?Glu?His?Asp?Gln?Phe?Ala?Glu?Thr?Leu?Lys?Gln?Gln
50??????????????????55??????????????????60
Gly?Ala?Glu?Val?Leu?Tyr?Leu?Glu?Lys?Leu?Thr?Ala?Glu?Ala?Leu?Asp
65??????????????????70??????????????????75??????????????????80
Asp?Ala?Leu?Val?Arg?Glu?Gln?Phe?Ile?Asp?Glu?Leu?Leu?Thr?Glu?Ser
85??????????????????90??????????????????95
Lys?Ala?Asp?Ile?Asn?Gly?Ala?Tyr?Asp?Arg?Leu?Lys?Glu?Phe?Leu?Leu
100?????????????????105?????????????????110
Thr?Phe?Asp?Ala?Asp?Ser?Met?Val?Glu?Gln?Val?Met?Ser?Gly?Ile?Arg
115?????????????????120?????????????????125
Lys?Asn?Glu?Leu?Glu?Arg?Glu?Lys?Lys?Ser?His?Leu?His?Glu?Leu?Met
130?????????????????135?????????????????140
Glu?Asp?His?Tyr?Pro?Phe?Tyr?Leu?Asp?Pro?Met?Pro?Asn?Leu?Tyr?Phe
145?????????????????150?????????????????155?????????????????160
Thr?Arg?Asp?Pro?Ala?Ala?Ala?Ile?Gly?Ser?Gly?Leu?Thr?Ile?Asn?Lys
165?????????????????170?????????????????175
Met?Lys?Glu?Pro?Ala?Arg?Arg?Arg?Glu?Ser?Leu?Phe?Met?Arg?Tyr?Ile
180?????????????????185?????????????????190
Ile?Asn?His?His?Pro?Arg?Phe?Lys?Gly?His?Glu?Ile?Pro?Val?Trp?Leu
195?????????????????200?????????????????205
Asp?Arg?Asp?Phe?Lys?Phe?Asn?Ile?Glu?Gly?Gly?Asp?Glu?Leu?Val?Leu
210?????????????????215?????????????????220
Asn?Glu?Glu?Thr?Val?Ala?Ile?Gly?Val?Ser?Glu?Arg?Thr?Thr?Ala?Gln
225?????????????????230?????????????????235?????????????????240
Ala?Ile?Glu?Arg?Leu?Val?Arg?Asn?Leu?Phe?Gln?Arg?Gln?Ser?Arg?Ile
245?????????????????250?????????????????255
Arg?Arg?Val?Leu?Ala?Val?Glu?Ile?Pro?Lys?Ser?Arg?Ala?Phe?Met?His
260?????????????????265?????????????????270
Leu?Asp?Thr?Val?Phe?Thr?Met?Val?Asp?Arg?Asp?Gln?Phe?Thr?Ile?His
275?????????????????280?????????????????285
Pro?Ala?Ile?Gln?Gly?Pro?Glu?Gly?Asp?Met?Arg?Ile?Phe?Val?Leu?Glu
290?????????????????295?????????????????300
Arg?Gly?Lys?Thr?Ala?Asp?Glu?Ile?His?Thr?Thr?Glu?Glu?His?Asn?Leu
305?????????????????310?????????????????315?????????????????320
Pro?Glu?Val?Leu?Lys?Arg?Thr?Leu?Gly?Leu?Ser?Asp?Val?Asn?Leu?Ile
325?????????????????330?????????????????335
Phe?Cys?Gly?Gly?Gly?Asp?Glu?Ile?Ala?Ser?Ala?Arg?Glu?Gln?Trp?Asn
340?????????????????345?????????????????350
Asp?Gly?Ser?Asn?Thr?Leu?Ala?Ile?Ala?Pro?Gly?Val?Val?Val?Thr?Tyr
355?????????????????360?????????????????365
Asp?Arg?Asn?Tyr?Ile?Ser?Asn?Glu?Cys?Leu?Arg?Glu?Gln?Gly?Ile?Lys
370?????????????????375?????????????????380
Val?Ile?Glu?Ile?Pro?Ser?Gly?Glu?Leu?Ser?Arg?Gly?Arg?Gly?Gly?Pro
385?????????????????390?????????????????395?????????????????400
Arg?Cys?Met?Ser?Met?Pro?Leu?Tyr?Arg?Glu?Asp?Val?Lys
405?????????????????410
<210>13
<211>408
<212>PRT
＜213〉enterococcus faecalis (Enterococcus faecalis)
<400>13
Met?Ser?His?Pro?Ile?Asn?Val?Phe?Ser?Glu?Ile?Gly?Lys?Leu?Lys?Thr
1???????????????5???????????????????10??????????????????15
Val?Met?Leu?His?Arg?Pro?Gly?Lys?Glu?Leu?Glu?Asn?Leu?Met?Pro?Asp
20??????????????????25??????????????????30
Tyr?Leu?Glu?Arg?Leu?Leu?Phe?Asp?Asp?Ile?Pro?Phe?Leu?Glu?Lys?Ala
35??????????????????40??????????????????45
Gln?Ala?Glu?His?Asp?Ala?Phe?Ala?Glu?Leu?Leu?Arg?Ser?Lys?Asp?Ile
50??????????????????55??????????????????60
Glu?Val?Val?Tyr?Leu?Glu?Asp?Leu?Ala?Ala?Glu?Ala?Leu?Ile?Asn?Glu
65??????????????????70??????????????????75??????????????????80
Glu?Val?Arg?Arg?Gln?Phe?Ile?Asp?Gln?Phe?Leu?Glu?Glu?Ala?Asn?Ile
85??????????????????90??????????????????95
Arg?Ser?Glu?Ser?Ala?Lys?Glu?Lys?Val?Arg?Glu?Leu?Met?Leu?Glu?Ile
100?????????????????105?????????????????110
Asp?Asp?Asn?Glu?Glu?Leu?Ile?Gln?Lys?Ala?Ile?Ala?Gly?Ile?Gln?Lys
115?????????????????120?????????????????125
Gln?Glu?Leu?Pro?Lys?Tyr?Glu?Gln?Glu?Phe?Leu?Thr?Asp?Met?Val?Glu
130?????????????????135?????????????????140
Ala?Asp?Tyr?Pro?Phe?Ile?Ile?Asp?Pro?Met?Pro?Asn?Leu?Tyr?Phe?Thr
145?????????????????150?????????????????155?????????????????160
Arg?Asp?Asn?Phe?Ala?Thr?Met?Gly?His?Gly?Ile?Ser?Leu?Asn?His?Met
165?????????????????170?????????????????175
Tyr?Ser?Val?Thr?Arg?Gln?Arg?Glu?Thr?Ile?Phe?Gly?Gln?Tyr?Ile?Phe
180?????????????????185?????????????????190
Asp?Tyr?His?Pro?Arg?Phe?Ala?Gly?Lys?Glu?Val?Pro?Arg?Val?Tyr?Asp
195?????????????????200?????????????????205
Arg?Ser?Glu?Ser?Thr?Arg?Ile?Glu?Gly?Gly?Asp?Glu?Leu?Ile?Leu?Ser
210?????????????????215?????????????????220
Lys?Glu?Val?Val?Ala?Ile?Gly?Ile?Ser?Gln?Arg?Thr?Asp?Ala?Ala?Ser
225?????????????????230?????????????????235?????????????????240
Ile?Glu?Lys?Ile?Ala?Arg?Asn?Ile?Phe?Glu?Gln?Lys?Leu?Gly?Phe?Lys
245?????????????????250?????????????????255
Asn?Ile?Leu?Ala?Phe?Asp?Ile?Gly?Glu?His?Arg?Lys?Phe?Met?His?Leu
260?????????????????265?????????????????270
Asp?Thr?Val?Phe?Thr?Met?Ile?Asp?Tyr?Asp?Lys?Phe?Thr?Ile?His?Pro
275?????????????????280?????????????????285
Glu?Ile?Glu?Gly?Gly?Leu?Val?Val?Tyr?Ser?Ile?Thr?Glu?Lys?Ala?Asp
290?????????????????295?????????????????300
Gly?Asp?Ile?Gln?Ile?Thr?Lys?Glu?Lys?Asp?Thr?Leu?Asp?Asn?Ile?Leu
305?????????????????310?????????????????315?????????????????320
Cys?Lys?Tyr?Leu?His?Leu?Asp?Asn?Val?Gln?Leu?Ile?Arg?Cys?Gly?Ala
325?????????????????330?????????????????335
Gly?Asn?Leu?Thr?Ala?Ala?Ala?Arg?Glu?Gln?Trp?Asn?Asp?Gly?Ser?Asn
340?????????????????345?????????????????350
Thr?Leu?Ala?Ile?Ala?Pro?Gly?Glu?Val?Val?Val?Tyr?Asp?Arg?Asn?Thr
355?????????????????360?????????????????365
Ile?Thr?Asn?Lys?Ala?Leu?Glu?Glu?Ala?Gly?Val?Lys?Leu?Asn?Tyr?Ile
370?????????????????375?????????????????380
Pro?Gly?Ser?Glu?Leu?Val?Arg?Gly?Arg?Gly?Gly?Pro?Arg?Cys?Met?Ser
385?????????????????390?????????????????395?????????????????400
Met?Pro?Leu?Tyr?Arg?Glu?Asp?Leu
405
<210>14
<211>409
<212>PRT
＜213〉pure mellow wine breast genus bacillus (Lactobacillus sake)
<400>14
Met?Thr?Ser?Pro?Ile?His?Val?Asn?Ser?Glu?Ile?Gly?Lys?Leu?Lys?Thr
1???????????????5???????????????????10??????????????????15
Val?Leu?Leu?Lys?Arg?Pro?Gly?Lys?Glu?Val?Glu?Asn?Ile?Thr?Pro?Asp
20??????????????????25??????????????????30
Ile?Met?Tyr?Arg?Leu?Leu?Phe?Asp?Asp?Ile?Pro?Tyr?Leu?Pro?Thr?Ile
35??????????????????40??????????????????45
Gln?Lys?Glu?His?Asp?Gln?Phe?Ala?Gln?Thr?Leu?Arg?Asp?Asn?Gly?Val
50??????????????????55??????????????????60
Glu?Val?Leu?Tyr?Leu?Glu?Asn?Leu?Ala?Ala?Glu?Ala?Ile?Asp?Ala?Gly
65??????????????????70??????????????????75??????????????????80
Asp?Val?Lys?Glu?Ala?Phe?Leu?Asp?Lys?Met?Leu?Asn?Glu?Ser?His?Ile
85??????????????????90??????????????????95
Lys?Ser?Pro?Gln?Val?Gln?Ala?Ala?Leu?Lys?Asp?Tyr?Leu?Ile?Ser?Met
100?????????????????105?????????????????110
Ala?Thr?Leu?Asp?Met?Val?Glu?Lys?Ile?Met?Ala?Gly?Val?Arg?Thr?Asn
115?????????????????120?????????????????125
Glu?Ile?Asp?Ile?Lys?Ser?Lys?Ala?Leu?Ile?Asp?Val?Ser?Ala?Asp?Asp
130?????????????????135?????????????????140
Asp?Tyr?Pro?Phe?Tyr?Met?Asp?Pro?Met?Pro?Asn?Leu?Tyr?Phe?Thr?Arg
145?????????????????150?????????????????155?????????????????160
Asp?Pro?Ala?Ala?Ser?Met?Gly?Asp?Gly?Leu?Thr?Ile?Asn?Lys?Met?Thr
165?????????????????170?????????????????175
Phe?Glu?Ala?Arg?Gln?Arg?Glu?Ser?Met?Phe?Met?Glu?Val?Ile?Met?Gln
180?????????????????185?????????????????190
His?His?Pro?Arg?Phe?Ala?Asn?Gln?Gly?Ala?Gln?Val?Trp?Arg?Asp?Arg
195?????????????????200?????????????????205
Asp?His?Ile?Asp?Arg?Met?Glu?Gly?Gly?Asp?Glu?Leu?Ile?Leu?Ser?Asp
210?????????????????215?????????????????220
Lys?Val?Leu?Ala?Ile?Gly?Ile?Ser?Gln?Arg?Thr?Ser?Ala?Gln?Ser?Ile
225?????????????????230?????????????????235?????????????????240
Glu?Glu?Leu?Ala?Lys?Val?Leu?Phe?Ala?Asn?His?Ser?Gly?Phe?Glu?Lys
245?????????????????250?????????????????255
Ile?Leu?Ala?Ile?Lys?Ile?Pro?His?Lys?His?Ala?Met?Met?His?Leu?Asp
260?????????????????265?????????????????270
Thr?Val?Phe?Thr?Met?Ile?Asp?Tyr?Asp?Lys?Phe?Thr?Ile?His?Pro?Gly
275?????????????????280?????????????????285
Ile?Gln?Gly?Ala?Gly?Gly?Met?Val?Asp?Thr?Tyr?Ile?Leu?Glu?Pro?Gly
290?????????????????295?????????????????300
Asn?Asn?Asp?Glu?Ile?Lys?Ile?Thr?His?Gln?Thr?Asp?Leu?Glu?Lys?Val
305?????????????????310?????????????????315?????????????????320
Leu?Arg?Asp?Ala?Leu?Glu?Val?Pro?Glu?Leu?Thr?Leu?Ile?Pro?Cys?Gly
325?????????????????330?????????????????335
Gly?Gly?Asp?Ala?Val?Val?Ala?Pro?Arg?Glu?Gln?Trp?Asn?Asp?Gly?Ser
340?????????????????345?????????????????350
Asn?Thr?Leu?Ala?Ile?Ala?Pro?Gly?Val?Val?Val?Thr?Tyr?Asp?Arg?Asn
355?????????????????360?????????????????365
Tyr?Val?Ser?Asn?Glu?Asn?Leu?Arg?Gln?Tyr?Gly?Ile?Lys?Val?Ile?Glu
370?????????????????375?????????????????380
Val?Pro?Ser?Ser?Glu?Leu?Ser?Arg?Gly?Arg?Gly?Gly?Pro?Arg?Cys?Met
385?????????????????390?????????????????395?????????????????400
Ser?Met?Pro?Leu?Val?Arg?Arg?Lys?Thr
405

Claims (37)

1.一种包含通过连接基团与聚乙二醇共价相连的精氨酸脱亚胺酶的化合物，其中所述聚乙二醇的总重均分子量为从约1,000到约40,000，以及所述的连接基团选自琥珀酰亚胺基、酰胺基、酰亚胺基、氨基甲酸酯基团、酯基、环氧基、羧基、羟基、碳水化合物、酪氨酸基团、半胱氨酸基团、组氨酸基团及其组合。1. A compound comprising arginine deiminase covalently linked by a linking group to polyethylene glycol, wherein the total weight average molecular weight of the polyethylene glycol is from about 1,000 to about 40,000, and the The linking group is selected from succinimide group, amide group, imide group, carbamate group, ester group, epoxy group, carboxyl group, hydroxyl group, carbohydrate, tyrosine group, cysteine Amino acid groups, histidine groups and combinations thereof. 2.如权利要求1所述的化合物，其中所述的连接基团是琥珀酰亚胺基团。2. The compound of claim 1, wherein said linking group is a succinimide group. 3.如权利要求2所述的化合物，其中所述琥珀酰亚胺基团是琥珀酸琥珀酰亚胺酯、丙酸琥珀酰亚胺酯、羧甲基化琥珀酰亚胺、琥珀酰亚胺基琥珀酰胺，N-羟基琥珀酰亚胺或其组合。3. The compound of claim 2, wherein the succinimide group is succinimide succinate, succinimide propionate, carboxymethylated succinimide, succinimide hydroxysuccinamide, N-hydroxysuccinimide or combinations thereof. 4.如权利要求3所述的化合物，其中所述琥珀酰亚胺基团是琥珀酸琥珀酰亚胺酯、丙酸琥珀酰亚胺酯或其组合。4. The compound of claim 3, wherein the succinimide group is succinimidyl succinate, succinimidyl propionate, or a combination thereof. 5.如权利要求1所述的化合物，其中所述精氨酸脱亚胺酶来源于支原体属的微生物。5. The compound of claim 1, wherein the arginine deiminase is derived from a microorganism of the genus Mycoplasma. 6.如权利要求5所述的化合物，其中所述的微生物选自精氨酸支原体(Mycoplasnia arginini)、人型支原体(Mycoplasma hominus)、关节炎支原体(Mycoplasma arthritides)及其组合。6. The compound of claim 5, wherein the microorganism is selected from the group consisting of Mycoplasma arginini, Mycoplasma hominus, Mycoplasma arthritis and combinations thereof. 7.如权利要求1所述的化合物，其中所述的精氨酸脱亚胺酶来源于链球菌属的微生物。7. The compound of claim 1, wherein the arginine deiminase is derived from a microorganism of the genus Streptococcus. 8.如权利要求7所述的化合物，其中所述的微生物选自化脓链球菌(Steptococcus pyogenes)、肺炎链球菌(Steptococcus pneumoniae)及其组合。8. The compound of claim 7, wherein said microorganism is selected from the group consisting of Streptococcus pyogenes, Streptococcus pneumoniae and combinations thereof. 9.如权利要求1所述的化合物，其中所述的精氨酸脱亚胺酶来源于疏螺旋体属的微生物。9. The compound of claim 1, wherein said arginine deiminase is derived from a microorganism of the genus Borrelia. 10.如权利要求9所述的化合物，其中所述的微生物选自布氏疏螺旋体(Borrelia burgdorferi)、阿氏疏螺旋体(Borrelia afzelii)及其组合。10. The compound of claim 9, wherein the microorganism is selected from the group consisting of Borrelia burgdorferi, Borrelia afzelii and combinations thereof. 11.如权利要求1所述的化合物，其中所述的精氨酸脱亚胺酶来源于Qiardia属的微生物。11. The compound of claim 1, wherein the arginine deiminase is derived from a microorganism of the genus Qiardia. 12.如权利要求11所述的化合物，其中所述的微生物是Qiardiaintestinalis。12. The compound of claim 11, wherein said microorganism is Qiardia intestinalis. 13.如权利要求1所述的化合物，其中所述的精氨酸脱亚胺酶来源于梭状芽孢杆菌属的微生物。13. The compound of claim 1, wherein the arginine deiminase is derived from a microorganism of the genus Clostridium. 14.如权利要求13所述的化合物，其中所述的微生物是产气荚膜梭状芽孢杆菌(Clostridium perfringens)。14. The compound of claim 13, wherein said microorganism is Clostridium perfringens. 15.如权利要求1所述的化合物，其中所述的精氨酸脱亚胺酶来源于肠球菌属的微生物。15. The compound of claim 1, wherein the arginine deiminase is derived from a microorganism of the genus Enterococcus. 16.如权利要求15所述的化合物，其中所述的微生物是粪肠球菌(Enterococcus faecalis)。16. The compound of claim 15, wherein said microorganism is Enterococcus faecalis. 17.如权利要求1所述的化合物，其中所述的精氨酸脱亚胺酶来源于乳杆菌属的微生物。17. The compound of claim 1, wherein the arginine deiminase is derived from a microorganism of the genus Lactobacillus. 18.如权利要求17所述的化合物，其中所述的微生物是清酒乳芽孢杆菌(Lactobacillus sake)。18. The compound of claim 17, wherein the microorganism is Lactobacillus sake. 19.如权利要求1所述的化合物，其中所述的精氨酸脱亚胺酶来源于芽孢杆菌属的微生物。19. The compound of claim 1, wherein the arginine deiminase is derived from a microorganism of the genus Bacillus. 20.如权利要求19所述的化合物，其中所述的微生物是地衣芽孢杆菌(Bacillus licheniformis)。20. The compound of claim 19, wherein said microorganism is Bacillus licheniformis. 21.如权利要求1所述的化合物，其中所述的微生物选自肺炎支原体(Mycoplasma pneumoniae)、人型支原体(Mycoplasma hominus)、精氨酸支原体(Mycoplasma arginini)、化脓链球菌(Steptococcuspyogenes)、肺炎链球菌(Steptococcus pneumoniae)、布氏疏螺旋体(Borrelia burgdorferi)、阿氏疏螺旋体(Borrelia afzelii)、Qiardiaintestinalis、产气荚膜梭状芽孢杆菌(Clostridium perfringens)、地衣芽孢杆菌(Bacillus licheniformis)、粪肠球菌(Enterococcus faecalis)、清酒乳芽孢杆菌(Lactobacillus sake)及其组合。21. The compound of claim 1, wherein said microorganism is selected from Mycoplasma pneumoniae (Mycoplasma pneumoniae), Mycoplasma hominus (Mycoplasma hominus), Mycoplasma arginini (Mycoplasma arginini), Streptococcus pyogenes (Steptococcuspyogenes), pneumoniae Streptococcus pneumoniae, Borrelia burgdorferi, Borrelia afzelii, Qiardiaintestinalis, Clostridium perfringens, Bacillus licheniformis, fecal enteritis Enterococcus faecalis, Lactobacillus sake, and combinations thereof. 22.如权利要求1所述的化合物，其中所述的精氨酸脱亚胺酶共价连接于约7到约15个聚乙二醇分子。22. The compound of claim 1, wherein said arginine deiminase is covalently linked to about 7 to about 15 molecules of polyethylene glycol. 23.如权利要求22所述的化合物，其中所述的精氨酸脱亚胺酶共价连接于约9到约12个聚乙二醇分子。23. The compound of claim 22, wherein said arginine deiminase is covalently linked to about 9 to about 12 molecules of polyethylene glycol. 24.如权利要求1所述的化合物，其中所述的聚乙二醇的总重均分子量为从约10,000到约30,000。24. The compound of claim 1, wherein said polyethylene glycol has a total weight average molecular weight of from about 10,000 to about 30,000. 25.一种延长精氨酸脱亚胺酶的循环半衰期的方法，该方法包括通过连接基团把所述的精氨酸脱亚胺酶与聚乙二醇共价相连来修饰所述的精氨酸脱亚胺酶，其中所述的聚乙二醇的总重均分子量为从约1,000到约4,000，以及所述的连接基团选自琥珀酰亚胺基、酰胺基、酰亚胺基、氨基甲酸酯基团、酯基、环氧基、羧基、羟基、碳水化合物、酪氨酸基团、半胱氨酸基团、组氨酸基团及其组合。25. A method for extending the circulating half-life of arginine deiminase, the method comprising covalently linking said arginine deiminase to polyethylene glycol via a linking group to modify said arginine deiminase Amino acid deiminase, wherein the total weight average molecular weight of the polyethylene glycol is from about 1,000 to about 4,000, and the linking group is selected from the group consisting of succinimide, amide, imide , carbamate groups, ester groups, epoxy groups, carboxyl groups, hydroxyl groups, carbohydrates, tyrosine groups, cysteine groups, histidine groups, and combinations thereof. 26.一种增强精氨酸脱亚胺酶抗肿瘤活性的方法，该方法包括通过连接基团把所述的精氨酸脱亚胺酶与聚乙二醇共价相连来修饰所述的精氨酸脱亚胺酶，其中所述的聚乙二醇分子的总重均分子量为从约1,000到约4,000，以及所述的连接基团选自琥珀酰亚胺基、酰胺基、酰亚胺基、氨基甲酸酯基团、酯基、环氧基、羧基、羟基、碳水化合物、酪氨酸基团、半胱氨酸基团、组氨酸基团及其组合。26. A method for enhancing the anti-tumor activity of arginine deiminase, the method comprising covalently linking the arginine deiminase to polyethylene glycol through a linking group to modify the arginine deiminase Amino acid deiminase, wherein the total weight average molecular weight of the polyethylene glycol molecule is from about 1,000 to about 4,000, and the linking group is selected from the group consisting of succinimide, amide, imide groups, carbamate groups, ester groups, epoxy groups, carboxyl groups, hydroxyl groups, carbohydrates, tyrosine groups, cysteine groups, histidine groups, and combinations thereof. 27.一种治疗患者的肿瘤的方法，该方法包括对所述患者施用如权利要求1所述的化合物。27. A method of treating a tumor in a patient, the method comprising administering the compound of claim 1 to said patient. 28.如权利要求27所述的方法，其中所述的肿瘤是黑素瘤。28. The method of claim 27, wherein said tumor is melanoma. 29.如权利要求28所述的方法，其中所述的聚乙二醇的总重均分子量约为20,000。29. The method of claim 28, wherein said polyethylene glycol has a total weight average molecular weight of about 20,000. 30.如权利要求28所述的方法，其中所述的连接基团是琥珀酰亚胺基团。30. The method of claim 28, wherein said linking group is a succinimide group. 31.如权利要求30所述的方法，其中所述琥珀酰亚胺基团是琥珀酸琥珀酰亚胺酯、丙酸琥珀酰亚胺酯、羧甲基化琥珀酰亚胺、琥珀酰亚胺基琥珀酰胺、N-羟基琥珀酰亚胺或其组合。31. The method of claim 30, wherein the succinimide group is succinimidyl succinate, succinimidyl propionate, carboxymethylated succinimide, succinimide hydroxysuccinamide, N-hydroxysuccinimide, or combinations thereof. 32.如权利要求27所述的方法，其中所述的肿瘤是肝细胞瘤。32. The method of claim 27, wherein said tumor is hepatoma. 33.如权利要求32所述的方法，其中所述聚乙二醇的总重均分子量约为5,000。33. The method of claim 32, wherein the polyethylene glycol has a total weight average molecular weight of about 5,000. 34.如权利要求32所述的方法，其中所述的连接基团是琥珀酰亚胺基团。34. The method of claim 32, wherein said linking group is a succinimide group. 35.如权利要求34所述的方法，其中所述琥珀酰亚胺基团是琥珀酸琥珀酰亚胺酯、丙酸琥珀酰亚胺酯、羧甲基化琥珀酰亚胺、琥珀酰亚胺基琥珀酰胺、N-羟基琥珀酰亚胺或其组合。35. The method of claim 34, wherein the succinimide group is succinimidyl succinate, succinimidyl propionate, carboxymethylated succinimide, succinimide hydroxysuccinamide, N-hydroxysuccinimide, or combinations thereof. 36.如权利要求27所述的方法，其中所述的肿瘤是肉瘤。36. The method of claim 27, wherein said tumor is a sarcoma. 37.一种治疗和抑制患者肿瘤转移的方法，该方法包括对所述患者施用如权利要求1所述的化合物。37. A method of treating and inhibiting tumor metastasis in a patient, the method comprising administering the compound of claim 1 to said patient.
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