CN1768138A

CN1768138A - Method for producing recombinant proteins in micro-organisms

Info

Publication number: CN1768138A
Application number: CNA038033267A
Authority: CN
Inventors: 鲁迪·舒希洛; 汉斯·克里斯琴·科廷; 汉斯·贡特尔·加森; 马丁·希尔斯; 拉尔夫·帕斯特纳克
Original assignee: N Zyme Biotec GmbH
Current assignee: N Zyme Biotec GmbH
Priority date: 2002-02-06
Filing date: 2003-02-06
Publication date: 2006-05-03
Also published as: EP1472346A2; AU2003210137A1; AU2003210137A8; WO2003066842A3; WO2003066842A2; MXPA04007585A; CA2475277A1; JP2005525798A

Abstract

The invention relates to a method for producing a recombinant functional plasminogen in micro-organisms, and to a method for identifying plasminogen activators. The nucleic acid sequence coding for the functional part of the plasminogen is fused with a nucleic acid molecule coding for at least one signal peptide. The nucleic acid molecule coding for the plasminogen and the nucleic acid molecule coding for the signal peptide are combined with codons for interfaces of proteases which ensure the separation of the signal peptide. The recombinant plasminogen or the corresponding plasmine is suitable for treating wounds which are slow to heal or not healing, by application of the enzyme in an appropriate formulation.

Description

In microorganism, produce the method for recombinant protein

Technical field

The present invention relates in microorganism, produce the method for recombinant protein.More specifically, the method that the present invention relates in microorganism, to produce the method for recombination function Profibrinolysin and identify plasminogen activator.

Background technology

Human fiber's protein dissolution system has comprised a nucleus-plasmin (Pm is hereinafter to be referred as plasmin).Plasmin on the one hand can fibrin degradation, can activate matrix metalloproteinase (MMPs) and somatomedin on the other hand, and activates matrix metalloproteinase (MMPs) and somatomedin plays a role in degradation of extracellular matrix and wound healing jointly.

Plasmin derives from its precursor molecule-plasmin former (hereinafter to be referred as Profibrinolysin).Up to now, oneself knows that two kinds of physiological activator of Profibrinolysin (are also referred to as plasminogen activator, PA).They be tissue-type plasminogen activator (tissue-type PA, t-PA) and urokinase type plasminogen activator (urokinase type PA, u-PA).In addition, this cover system is regulated by a cover proteinase inhibitor such as α 2-antiplasmin.Profibrinolysin and plasmin have very important biological function, and these two kinds of biological functions are directly related with two kinds of different activator respectively.

The approach of so-called t-PA mediation is responsible for the running balance of control agent inner fibrin, and the approach of u-PA mediation cell move and tissue remodeling aspect effect outstanding.This function shows particularly outstandingly when suffering from chronic, disunion sexual trauma when the u-PA deficient mice.The same state of an illness can appear in Profibrinolysin, t-PA and u-PA the gene mouse of inactivation respectively.In addition, particularly owing to thrombosis and organ failure, the life-span of laboratory animal obviously shortens.About the general introduction of Profibrinolysin/plasmin system, in the book that Desire Collen publishes, described to some extent (" thrombosis and hemostasis (Thrombosis and Haemostasis) ", 82,1999 (1)).

The plasmin therapy is applicable to heart attack or paralytic's treatment, in this case, the solution fibrin grumeleuse is that survival is necessary fast, therefore, described plasmin therapy is the another kind of mode except using plasminogen activator treatment patient, and plasminogen activator has only been facilitated the hydrolysis of fibrin clot indirectly.

The above-mentioned mouse disease model of mentioning has shown that plasmin still is a kind of potential medicine, is used for treating the disunion sexual trauma or the slow wound that only heals.

Usually the t-PA plasminogen activation only appears under the situation of scleroproein existence, i.e. after blood cascade is condensed and finished.Under the situation that lacks substrate, plasmin is always suppressed by α 2-antiplasmin immediately.Generally acknowledge when plasmin to be attached on the scleroproein, and thus can activation fiber albumen clot dissolution the time, this restraining effect obviously weakens.

Therefore because in heart attack or apoplexy case, thrombus normally patient is survived necessaryly, has used different Profibrinolysins to activate scheme in the treatment.For example, the injection streptokinase makes blood vessel logical more fast.Therefore, the streptokinase plasminogen activation is not to be based upon on the proteolysis activatory basis, but has formed mixture that then, this mixture activates other Profibrinolysin molecule and makes it change plasmin into, and described streptokinase is a kind of bacterioprotein.

The further research that urokinase treatment is used is thought that as streptokinase, urokinase can not distinguished Profibrinolysin and the free Profibrinolysin that is attached on the scleroproein on the molecular level.Therefore, developed recombinant human t-PA, in clinical study, confirmed that recombinant human t-PA effect is better than streptokinase.But the discovery on this diagnostics fails to be studies confirm that by other.

Have vital role in the recombinant protein that the production system of utilizing the method for molecular genetics to set up is used in producing modern treatment, and the importance that plasminogen activator, recombinant single chain urokinase-PA that reorganization produces waits such as rt-PA (and different derivatives) and the staphylokinase of recombinating have highlighted described production system just.

Profibrinolysin is the precursor molecule of fibrinoclase.CDNA (Malinowski etc., Biochemistry, 23,1984 (12) of human plasminogen in scientific literature, have been delivered; Forsgren etc., FEBS Lett.213,1987 (2)) and comprise the gene (Petersen etc., J.Biol.Chem., 265,1990 (3)) of non-coding intron.

Human plasminogen (hPg) is the proenzyme as the plasmin of serine protease, and the glycoprotein that it is made up of 791 amino acid whose polypeptide chains, molecular weight are 92,000, and theoretical iso-electric point is 7.1, and the glycosylation ratio is 2% (Collen, 1999, (1)).Generate Profibrinolysin in liver, plasma concentration is approximately 200mg/l[1.5-2 μ M (every liter of micromole)].

This molecule is divided into 7 structural domains, comprises preceding activating peptide (Glu-1～Lys-77), homeologous five the ring cake structure territories (Kringle domain) and the proteolytic enzyme structural domain (Val-562～Asn-791 with catalytic activity of N-terminal; Collen, 1999 (1)).The catalytic triplet configuration is consistent with the structure of all serine protease, all is made of following amino acid: His-603, Asp-646 and Ser-741.Ring cake structure territory 1 is the proteic recognition sequence of Profibrinolysin binding fiber (Petersen etc., 1990 (3)), also is Profibrinolysin and different cell surface receptor bonded recognition sequences.

In posttranslational modification, two essential glycosylation site Asn-289 and Thr-346 are arranged in ring cake structure territory 3, its function particularly important (have respectively by different proteolytic enzyme and streptokinase activated ability, also have receptor-binding characteristic) for Profibrinolysin.According to described modification, distinguish the Profibrinolysin of two kinds of principal modes:

-Profibrinolysin I has above-mentioned glycosylation characteristic

It is glycosylation modified that-Profibrinolysin II lacks Asn-289.

Another glycosylation site is amino acid Ser-248.Amino acid Ser-578 can exist with phosphorylation form.

In vivo, the proteolysis of Profibrinolysin between amino acid Arg-561 and Val-562 cuts off and activates.Subsequently, another proteolysis enzymic hydrolysis activation occurs between Lys-77 and the Lys-78, forms Lys-78-hPg.Perhaps in Glu-Pg, described chemical bond also can directly be hydrolyzed at first.Activatory plasmin Lys-78-hPm is all connected by disulfide linkage.Therefore, the heavy chain of hPm (1/78-561) is responsible for and substrate interaction, described substrate such as Fibrinogen and scleroproein.The light chain (562-791) that derives from C-terminal is the subunit with catalytic activity.

Known from document, the method for the scleroproein binding domains of recombinant production Profibrinolysin in pichia pastoris phaff (Pichia pastoris), its output are 17mg/l (Duman etc., Biotechnol Appl Biochem.28; 39-45,1998 (4)).The glycosylation site of this structural domain of author's susceptible of proof (ring cake 1-4).The production (Guan etc., Sheng Wu Gong Cheng Xue Bao, 17,2001 (5)) of ring cake 4 and 5 these two structural domains of human plasminogen described in another quoted passage.The purpose of this work is that evaluation can suppress the structural domain of endothelial cell growth.

Yet the Profibrinolysin structural domain by above-mentioned two work groups recombinant production in pichia pastoris phaff does not all contain the catalyst structure domain that physiological function is had decisive significance.

People such as Gonzalez-Gronow (Biochimica et Biophysica Acta, 1039,1990 (6)) have compared the expression of recombinant human plasminogen in intestinal bacteria (Escherichia coli) and COS cell (a kind of ape kidney cell line) mutually.Microorganisms producing in intestinal bacteria is failed, and it is insufficient that the author is attributed to glycosylation to it.Can successfully produce peptide chain, yet it is a kind of form that can not be activated, promptly use activator (urokinase and t-PA) to handle after, can not produce the activatory plasmin.

The potein deficiency glycosylation, will cause the important physiological function defective relevant (not detecting enzymic activity) with activation capacity, and the important physiological function defective (Gonzalez-Gronow etc. relevant with endotheliocyte identification, Biochimica et Biophysica Acta, 1039,1990 (6)).In addition, carry out posttranslational modification, there is remarkably influenced the half life of protein in mammalian with carbohydrate.

Yet this author has produced functional Profibrinolysin in the COS cell.Other authors have described the functional expression (Whitefleet-Smith etc., Arch.Biochem.Biophys., 271,1989 (7)) in insect cell.Yet, when using mammalian cell and insect cell, have following shortcoming: lose time, culture condition expends great amount of cost and the protein yield that obtains is low.In addition, in mammalian cell, because will be at cell inner expression albumen, and contain proteolytic enzyme in the tenuigenin, so be unsuitable for producing the more proenzyme of volume [Nilsen and Castellino, protein expression and purifying (Protein Expression and Purification), 16,1999 (8); Busby etc., J.Biol.Chem., 266,1991 (9)].Usually in baculovirus (baculovirus)/lepidopteran (lepidopteran) (insect cell) system, the expression output of this system is 3-10mg/ml.

At publication number is in the patent application of WO0250290, discloses the method for the functional Miniplasminogen of recombinant production (mini-plasminogen) and Microplasminogen (micro-plasminogen) in yeast.For this reason, the author has expressed catalytic structure domain gene (Miniplasminogen) that contains ring cake structure territory or the human plasminogen (Microplasminogen) that does not contain ring cake structure territory in the pichia pastoris phaff as host organisms.The purifying Miniplasminogen and the Microplasminogen of recombinating like this and producing respectively subsequently, and be processed into little plasmin and fento lyase respectively, and proved their activity by experimentation on animals.It is said that for Miniplasminogen the output of recombinant protein is 100mg/l; For Microplasminogen, the output of recombinant protein is 3mg/l.Yet the expression amount that makes the recombinant protein product is higher is more difficult, and this has obtained confirmation in publication number is the patent application of WO0250290, in described patent application, and the output of Miniplasminogen and Microplasminogen 2 orders of magnitude that obviously descended.The example of the embodiment of not mentioned any expression long segment Profibrinolysin varient, described long segment Profibrinolysin varient such as Methionin Profibrinolysin (Lys-plasminogen) or L-glutamic acid Profibrinolysin (Glu-plasminogen).

The still unexposed degree that can implement to those skilled in the art of the method for the functional Profibrinolysin of recombinant production in microorganism.

Summary of the invention

Therefore, the objective of the invention is to produce the functional human Profibrinolysin, and further make it be processed into plasmin with catalytic activity with cheap method.

According to claim 1, the method for recombinant production Profibrinolysin has been finished described purpose in microorganism.In independent claim, mentioned more solution.Dependent claims has reflected embodiment preferred.

Wonderful discovery is that production recombination function L-glutamic acid Profibrinolysin or Methionin Profibrinolysin are possible in microorganism.Studies show that further that on this basis reorganization Microplasminogen, Miniplasminogen, Methionin Profibrinolysin and L-glutamic acid Profibrinolysin can reach beyond thought high expression level amount.

Theme of the present invention is to adopt the molecular genetics method, clone's Profibrinolysin gene in expression vector, described Profibrinolysin gene is preferably Microplasminogen and Miniplasminogen gene, more preferably L-glutamic acid Profibrinolysin or Methionin Profibrinolysin gene, or every kind of function varient of aforementioned gene; Produce functional Profibrinolysin with reorganization, be preferably the functional human Profibrinolysin.In addition, the invention describes a kind of authentication method of proteolytic enzyme, described proteolytic enzyme catalysis Profibrinolysin changes plasmin into.The Profibrinolysin and the plasmin that utilize the present invention to produce respectively can not polluted by animal albumen or virus, and described pollution appears at usually from people, ox and other Mammals to be carried out the separating process, and can cause patient's untoward reaction.

The invention is characterized in a kind of method of producing recombinant protein, this method may further comprise the steps at least: the nucleotide sequence of the funtion part of the Profibrinolysin peptide section of a) will encoding at least merges with the nucleotide sequence of the segment signal peptide of encoding at least, be cleavable signal peptide, the nucleotide sequence of described encoding function Profibrinolysin peptide section and at least the nucleotide sequence of coded signal peptide by the restriction enzyme site codon coupling of proteolytic enzyme.The recombinant production system is the manufacture of therapeutic protein product more and more.Because cost factor will be striven in microorganism, particularly produce this recombinant products in the bacterial body.Described system has following advantage: except the product relative low price, protein yield can reach the g/l level, and recombinant protein can be not contaminated, and this pollution is meant deleterious virus of patient or the albumen as the Protein virus.Because the bacterium production system can not generate correct folding albumen usually, therefore, except the albumen to false folding carry out external folding again, usually in eukaryotic cell system, produce, described eukaryotic cell system is yeast, insect cell or mammalian cell for example.The eucaryotic cell strain and the clone that are used to produce provide favourable condition, and promptly they can be used to produce glycosylated protein.But especially the recombinant protein product cost in insect cell or mammalian cell source is high, and output is very low usually.In addition, they also have shortcoming, can be by harmful virus and protein contamination.Described contaminated situation can not appear to be used under the eukaryotic microorganisms situation.Eukaryotic microorganisms is cultivated required plant and instrument and the required unit affinity of microbial culture, can not occur by the situation of mammalian virus and protein contamination, and protein yield can reach the g/l level.Particularly suitable a kind of eucaryon host organism is branch of zymic, is preferably Ascomycota (Ascomycota).More preferably Saccharomycotina, especially yeast (Saccharomycetes) guiding principle, wherein yeast (Saccharomycetales) order is particularly important.According to more superior embodiment, the host should be yeast (Saccharomycetaceae) section, especially Pichia (Pichia).The yeast saccharomyces cerevisiae (Saccharomycescerevisiae) that the preferred exemplary eukaryotic microorganisms of the present invention is used always for the baker, other for example Candida (Candida), the pichia pastoris phaff (Pichia pastoris) that belongs to the methyl alcohol nutritious yeast, pichia spp of methyl alcohol trophicity (Pichiamethanolica) and multiple-shaped nuohan inferior yeast (Hansenula polymorpha), or the filamentous fungus of Aspergillus (Aspergillus) such as aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae) and Aspergillus nidulans (Aspergillus nidulans).Particularly preferably be pichia pastoris phaff.

Production method another feature of described recombinant products is, the nucleic acid molecule of the funtion part of the Profibrinolysin of encoding at least is integrated into the expression vector that uses in these microorganisms, the nucleic acid molecule of these optimized encoding human plasminogens, merge with the nucleic acid molecule of at least one signal peptide of coding [be preferably one section propetide former (prepropeptide), be preferred for being transported to endoplasmic reticulum]; The codon at proteolytic enzyme point of contact inserts between described two nucleic acid molecule, can be in host, and cutoff signal sequence or propetide are former.The preferred nucleic acid molecule that uses is the nucleic acid molecule of coding human plasminogen.Except the nucleic acid molecule of coding human plasminogen, also can use those codings to derive from the nucleic acid molecule of other Mammals Profibrinolysin.Like this, just can produce various mammiferous Profibrinolysins.The method according to this invention can form the human plasminogen of reorganization by overexpression, if desired, it can be advanced substratum by secretion, and the method that oneself knows according to present those of skill in the art, it is separated by centrifugal, filtration or precipitation and host cell in the substratum, and carry out protein purification, and do not need complicated lysis process.Can Profibrinolysin be activated by proteolytic enzyme becomes plasmin, and this proteolytic enzyme has the ability that Profibrinolysin is processed as the plasmin with catalytic activity.

The following term of Shi Yonging is defined as in the present invention:

" recombinant method for production " looks like, and in suitable host, nucleotide sequence (being preferably dna sequence dna) is expressed and generated peptide or protein, and described nucleotide sequence derives from a clone, and the fusion product of single nucleic acid fragment.

" clone " as herein described should comprise all known cloning process of the prior art.Yet, can not describe described method in detail, because they belong to the conspicuous common technology of those skilled in the art.

" expressing in appropriate expression system " as herein described should comprise all known expression methods of the prior art, the expression method of especially mentioning in claims.

In " functional Profibrinolysin peptide section part " this term, be construed as part Profibrinolysin or the Profibrinolysin peptide section (plasminogen-peptide) that to finish the relevant biological function of Profibrinolysin.These relevant biological functions have at least and can be activated the ability that becomes plasmin by plasminogen activator, and described activator for example is tissue plasminogen activator, urokinase, vampire (vampire-bat) plasminogen activator, streptokinase, staphylokinase (hereinafter to be referred as staphylokinase) and the Pla albumen that derives from Yersinia pestis (Yersinia pestis) etc.; With proteoclastic ability, it is characterized in that hydrolysis of fibrin.Used term " plasminogen activator " in this specification sheets and embodiment, Ying Zhineng carry out proteolysis and can not carry out proteoclastic plasminogen activator.

In addition, the L-glutamic acid Profibrinolysin is interpreted as, and by activating peptide before the plasmin catalyze cleavage (preactivation peptide), described L-glutamic acid Profibrinolysin has the ability that is formed to the Methionin Profibrinolysin.

After Profibrinolysin is attached to scleroproein, ln, Zeta protein, hyaluranectin, heparan sulfate proteoglycan, 4 Collagen Type VIs and other substrate, its activation capability can be brought up to 1000 times, equally, described activation capability belongs to biological function.

Should understand the plasmin that processing obtains behind the Profibrinolysin and should guarantee to have the relevant biological function of plasmin, comprising the degraded of following material: ln, Zeta protein, hyaluranectin and heparan sulfate proteoglycan; The activation of following material: precollagen enzyme, preceding matrix metalloproteinase, potential scavenger cell elastoser, prohormone and somatomedin, this somatomedin is TGF β-1 (potential transforming growth factor) for example, VEGF (vascular endothelial growth factor) or bFGF (basic fibroblast growth factor).

Another biological function of plasmin is by the ability that plasmin inhibitor suppressed, described plasmin inhibitor such as α ₂-antiplasmin and α ₂-macroglobulin.

Associated biomolecule according to plasmin is learned function, its function not only also comprises binding fiber albumen, ln, Zeta protein, hyaluranectin, heparan sulfate proteoglycan and 4 Collagen Type VIs, also comprises bind receptor such as α-enolase, annexin (annexin) II or the facultative factor (amphoterin).

At first, all Profibrinolysins of formation all are the L-glutamic acid Profibrinolysin forms of non-activity.This L-glutamic acid Profibrinolysin can be cut described preceding activating peptide and generate the Methionin Profibrinolysin by plasmin.The both can be organized plasminogen activator (in this case, only referring to above-mentioned proteolysis activator) and generate plasmin by the protease hydrolysis cutting, and this plasmin is made up of the subunit that disulfide linkage connects.Less subunit comprises proteolysis structural domain and phosphorylation site, and bigger subunit carries three glycosylation sites, and is responsible for being attached to scleroproein.The importance of glycosylation site further shows on the stability in the blood plasma.Profibrinolysin is by forming 1: 1 mixture with streptokinase or staphylokinase, and Profibrinolysin can change into a kind of enzyme with proteolytic activity again, and this endonuclease capable is processed as plasmin with Profibrinolysin.

In view of the above, functional Profibrinolysin is meant a kind of such Profibrinolysin, and it can be become the plasmin with proteolytic activity by the plasminogen activator effect.Further, functional Profibrinolysin preferably includes the scleroproein binding domains, and can comprise in three glycosylation sites one at least.

The minimum form of functional Profibrinolysin is Microplasminogen and Miniplasminogen, and big slightly form is the Methionin Profibrinolysin.The L-glutamic acid Profibrinolysin, it still comprises preceding activating peptide, also is functional Profibrinolysin.Yet conceivablely be, described zone can be left in the basket, and especially is present in the long-chain and does not have significantly to hinder the zone of described function (especially proteolysis function, scleroproein combined function).

It is apparent for those of skill in the art to make the multi-form Profibrinolysin that comprises functional catalyst structure domain (hereinafter referred to as the Profibrinolysin derivative).With regard to the function of having described, should understand after being activated the proteolysis characteristic that described Profibrinolysin varient has, described plasminogen activator such as streptokinase or urokinase by plasminogen activator.

-catalyst structure domain can comprise, disappearance and metathetical amino acid, or with other amino acid or peptide or albumen fusion.

-macrostructure territory can comprise all region intermediates (based on preceding Profibrinolysin sequence) from Glu20 to Arg580, and it can be activated by plasminogen activator and generate the activatory plasmin.

Below be the specific embodiment of the Methionin Profibrinolysin of three kinds of forms:

Varient 1:N end amino acid: Met88

Varient 2:N end amino acid: Lys97

Varient 3:N end amino acid: Val98

Preferably, the Profibrinolysin derivative more little than accordingly-, little-, short or about 1 to 50 amino acid of growing up of Lys-or Glu-Profibrinolysin; Or be feature to have replaced 1 to 10 amino acid preferably, in addition, described derivative shows by plasminogen activator activated characteristic.These specific little-, little-, between Lys-or Glu-Profibrinolysin and corresponding Profibrinolysin derivative, the homology of sequence (sequences match) surpasses 80%, preferably surpass 85%, more preferably surpass 90%, further preferably surpass 95%, especially preferably surpass 98%, further especially preferably surpass 99%.

Preferred Profibrinolysin derivative has following feature:

-catalyst structure domain can comprise, at least one disappearance, and/or at least one amino acid whose displacement, and/or with another amino acid at least or at least another peptide section or at least another albumen merge;

-macrostructure territory can comprise all region intermediates (based on preceding Profibrinolysin sequence) from Glu20 to Arg580, and it can be activated by plasminogen activator and generate the activatory plasmin;

-Profibrinolysin derivative is characterized in that the homology (sequences match) of aminoacid sequence surpasses 80%, preferably surpasses 85%, more preferably surpasses 90%, further preferably surpasses 95%, further especially preferably surpasses 99%.

" microorganism " comprises that all have possessed the life form of small personal feature.Therefore, it had both comprised eukaryotic microorganisms, had also comprised prokaryotic micro-organisms.Refer in particular to bacterium, yeast, fungi and virus.

" nucleic acid " comprises DNA and RNA, and both are all possible configuration, for example, and the nucleic acid of double chain form, the nucleic acid of single stranded form, their composition and traditional thread binding or cyclic nucleic acid.

Should understand the sequence that " signal sequence " is one section peptide chain, it can guarantee that other one section peptide chain-ordering enters or pass through microbial film, for example enters endoplasmic reticulum.Therefore, the example of described signal sequence can be propetide former (prepropeptide), propetide (prepeptide) or former peptide (propeptide).

" point of contact " is meant the site in the peptide chain-ordering, and described site provides the site for carry out following processing in host organisms: from another peptide section sequence cut away signal sequence, propetide is former or former peptide, or usually one section peptide section sequence is cut into two portions.

" the former nucleic acid of at least one signal peptide or propetide of encoding " is meant a nucleotide sequence, its encoded a peptide section or protein structure, and described peptide section or protein structure help other polypeptide to pass through microbial film, for example enter endoplasmic reticulum.

" primer " is meant a kind of initial oligonucleotide.The meaning is short chain, strand, oligomerization Yeast Nucleic Acid or thymus nucleic acid, its can with certain regional complementarity of single stranded nucleic acid molecule, and hybridization forms double-stranded with it.Free 3 ' in the two strands-C-terminal can be used as the substrate of archaeal dna polymerase, and as the starting point of whole strand being synthesized double-stranded polyreaction.Primer is specifically applied to PCR, i.e. polymerase chain reaction, described reaction dawn known to those skilled in the art.

" plasmid " is meant nucleic acid molecule, and it is not be integrated into karyomit(e), but appears in a lot of prokaryotic micro-organisms and some eukaryotic microorganisms, and length is approximately 2kb to surpassing 200kb.

" connection " refers to connect the terminal of two nucleic acid molecule or self connection by ligase enzyme, self connect and promptly refer to the connection finished through intramolecular ring-closure reaction, if wherein two of a linear DNA molecule strand ends can form base pair each other, both will dimerization.

" restriction endonuclease " is meant the enzyme that a bacterioid produces, and it can cut the phosphodiester bond in the specific base sequence of dna double chain molecule.

" electroporation " is a kind of method that nucleic acid enters cell that mediates.Therefore when recipient cell is exposed to nucleic acid solution, utilize the brief electrical pulse of high field intensity, make the local penetrating high-molecular weight molecule of cytolemma of the recipient cell that in suspension, grows to logarithmic phase.

Should understand " overexpression " and be meant, compare with the output of wild-type cell, the output of the functional Profibrinolysin of cells produce increases.Usually, overexpression is meant, when in cell, producing, the expression of exogenous gene amount account for host cell whole intracellular protein total amounts 1～40%.

Should understand " expression vector " and be meant such carrier, after this carrier entered proper host cell, it can transcribe the foreign gene that is cloned into this carrier, and translated with the mRNA (messenger RNA(mRNA)) that forms subsequently.Expression vector comprises control signal usually, and this control signal is essential in protokaryon or eukaryotic genetic expression.

In the present invention, preferably can be by the promotor of methanol induction such as AOX1-promotor, or special preferred group become second nature promotor such as YPT1-promotor or GAP-promotor, described promotor is used to control the expression of gene in the yeast, described yeast such as pichia pastoris phaff.Be preferably the GAP-promotor of composition especially.

" AOX1 " is the gene that is derived from the alcohol oxidase 1 of pichia pastoris phaff;

" GAP " is the gene that is derived from the Glyceraldehyde-3-phosphate desaturase of pichia pastoris phaff; And

" YPT1 " is the gene that is derived from the gtp binding protein of pichia pastoris phaff.

Proteic signal peptide by PHO-1, SUC-2, PHA-E or α-MF genes encoding is applied in the zymic secretory product usually.

" PH01 " is the acid phosphatase gene that is derived from pichia pastoris phaff;

" SUC-2 " is the secretion invertase gene that is derived from yeast saccharomyces cerevisiae;

" PHA-E " is the acid phosphatase gene that is derived from Phaseolus vulgaris Agglutinis;

" α-MF " is the α-hybridization factor gene that is derived from yeast saccharomyces cerevisiae.

Particularly preferably be the codon at proteolytic enzyme point of contact, reach Proteinase K ex2 or Ste13 and cut the codon at the point of contact of former peptide.A) carry out described connection particularly preferably in above-mentioned steps, described codon encoded K ex2 point of contact and two other Ste13 point of contact with codon.In a preferred embodiment of the present invention, the former nucleic acid molecule of coded signal peptide or propetide derives from yeast, especially derives from yeast saccharomyces cerevisiae.In a more preferred, relate to the former nucleic acid molecule of coded signal peptide or propetide, the signal peptide or the propetide of the α-factor of this nucleic acid molecule encoding yeast saccharomyces cerevisiae are former.The formed fusion product of describing in a) in above-mentioned steps is preferably by pcr amplification, and further preferably carries out suitable purifying.

At publication number is in the patent application of WO02/50290, discloses by being applicable to that zymic expression vector pPICZ α A reorganization produces Miniplasminogen and Microplasminogen, and it is former that described carrier contains the propetide of derivable AOX1-promotor and yeast α-factor.These less Profibrinolysin varients do not have (as Microplasminogen), or one (as Miniplasminogen) ring cake structure territory is only arranged.Expression vector pPICZ α A comprises Proteinase K ex2 and Ste13 point of contact.But in Miniplasminogen and Microplasminogen corresponding expression vector, the Ste13 point of contact is deleted.

The one group of promotor that is used for derivable expression system in the known yeast.Up to the present especially comprise, AOX1-promotor, AOX2, CUP1 (Koller A, Valesco J, SubramaniS., Yeast 2000:16 (7), 651-6), PH01 (EP0495208), HIS4 (US 4885242), FLD1 (Shen etc., Gene 1998:216 (1) is 93-10) with XYL1-promotor (Den Haan and Van Zyl, Appl.Microbiol.Biotechnol.2001:57 (4), 521-7).

The method of the AOX1-promotor by methanol induction can optionally directedly be produced allogenic albumen, and can be obtained the quantity of homogeneous.Inducing before foreign protein expresses, making host organisms can reach high stand density and the screening defective that in expressing foreign protein, do not take place.

Publication number is in the patent application of WO02/50290, under the control of AOX1-promotor, expressed less varient, opposite with described less varient, the L-glutamic acid Profibrinolysin of recombinant production and Methionin Profibrinolysin have comprised five all ring cake structure territories among the present invention, owing to following reason, make its recombinant production become complicated:

-host is when expressing foreign protein, because its growth defect may cause Expression element to be lost;

The proteolytic degradation of-expressing protein; With

-low yield.

Because described defective, be the production of unexposed L-glutamic acid Profibrinolysin and Methionin Profibrinolysin in the patent application of WO02/50290 at publication number.

In the present invention, these difficulties are solved especially in the following manner: recombinant protein has comprised signal peptide, Kex2 and at least one Ste13, preferred two Ste13 proteolytic enzyme point of contacts.Furthermore, in the embodiment preferred, supply is as the glycerine of another kind of carbon source, and its speed is 0.1 to 10ml/h, and more preferably 0.5 to 5ml/h, and more preferably 0.8 to 1.5ml/h.Substratum is the neutral buffer systems of pH7.0.Note guaranteeing sufficient oxygen supply.

In a preferred embodiment, should note: recombinant nucleic acid is not connected to 5 ' end of AOX1 gene, but has been connected to 5 ' end of the glyceraldehyde phosphate dehydrogenase gene that is derived from pichia pastoris phaff.Here, used composition, can not derivative promotor.Spendable in yeast activated composition promotor be GAP-promotor, YPT1-promotor (Sears etc., Yeast1998:14 (8), 783-90), TKL-promotor (Den Haan and Van Zyl, Appl.Microbiol.Biotechnol.2001:57 (4), 521-7), ACT-promotor (Kang etc., Appl.Microbiol.Biotechnol.2001:55 (6), 734-4 " and the PMA1-promotor (Yeast2000:16 (13), 1191-203).Preferred promotor is GAP-promotor and YPT1-promotor.Particularly preferred promotor is the GAP-promotor.

Opposite with inducible promoter, the promotor of composition has its defective, and promptly the foreign protein that will express continues the composition generation in the whole growth cycle.This is the unfavourable condition to host cell, especially shows as the host cell delayed growth.Because the advantage selective pressure, the host cell of having lost recombinant expressed element has growth vigor, and can the hypertrophy recombinant host cell.Like this, can occur in the mixed population of allos, and the mixed population of allos should avoid.Yet surprising discovery is that according to the preferred embodiment of the invention, composition GAP-promotor can produce higher output.

When using the AOX1-promotor, the output of Methionin Profibrinolysin after inducing 120 hours, can be obtained up to few 17U/l (units per liter) (=1.5mg/l), more preferably 120U/l (=11mg/l), more preferably 180U/l (=16mg/l), further preferably 200U/l (=18mg/l), again preferably 220U/l (=20mg/l), again further preferably 240U/l (=22mg/l), especially preferably 260U/l (=24mg/l), more special preferably 280U/l (=25.5mg/l); Using the composition promotor, particularly during the GAP-promotor, output significantly raises.

In a preferred embodiment, the promotor of composition such as GAP-promotor and the coupling of nucleic acid operability, described nucleic acid encoding is the funtion part of Profibrinolysin sequence at least, and merge with the nucleotide sequence of at least one segment signal peptide of coding, the nucleotide sequence of described encoding function Profibrinolysin and the restriction enzyme site codon coupling of the nucleotide sequence usefulness proteolytic enzyme of signal peptide at least of encoding, this proteolytic enzyme is used for cleavable signal peptide.

In particularly preferred embodiments, the promotor of composition such as GAP-promotor, operability coupling control is little-, little-, Lys-or Glu-Profibrinolysin nucleotide sequence, this sequence merges with the nucleotide sequence that is derived from the zymic signal peptide.

This can regard a wonderful discovery as, and according to the preferred embodiments of the invention, composition GAP-promotor can reach the output that is higher than 10 times (see embodiment 7c, the production of Methionin Profibrinolysin, 1375U/l can be scaled 125mg/l).In a further preferred embodiment, supply is as the glycerine of another kind of carbon source, and speed is 0.1 to 10ml/h (milliliter/hour), and more preferably 0.5 to 5ml/h, and more preferably 0.8 to 1.5ml/h, and substratum is the neutral buffer systems of pH=7.0.Therefore, speed of growth μ [l/h] is between 0.002 and 0.10, preferably between 0.004 and 0.020, further preferably between 0.008 and 0.010.

In using the GAP-promotor, after fermentation continues 250 hours, the output of the Methionin Profibrinolysin that obtains be at least 660U/l (=60mg/l), be preferably 1000U/l (=91mg/l), be preferably again 1500U/l (=136mg/l), more preferably 2000U/l (=182mg/ml), be preferably especially 2500U/l (=227mg/l), further be preferably especially 2750U/l (=250mg/l).

Therefore, reorganization Miniplasminogen and Microplasminogen product can obtain higher expression amount.The output of Miniplasminogen between the 2g, is preferably 300mg/l-1.5g/l between every liter of 100mg, and more preferably 400mg/l-1g/l further is preferably 500mg/l-800mg/l, is preferably 600mg/l-700mg/l especially.The output of Microplasminogen is higher than 10% of small-sized Profibrinolysin output at least.Compared to Methionin Profibrinolysin recombinant products, the output of the L-glutamic acid Profibrinolysin recombinant products of acquisition is low slightly.

Method of the present invention, be applicable to produce little-, little-, Lys-and Glu-Profibrinolysin.Therefore, preferred embodiment also concentrate on reorganization produce little-, little-, on Lys-and the Glu-Profibrinolysin, the above is little at expression vector-, little-, Lys-and Glu-Profibrinolysin all with signal sequence or preceding former sequence (prepro sequence) coupling, these carriers have comprised composition promotor such as GAP-promotor.In a further preferred embodiment, signal sequence is by former composition of propetide of a factor of signal peptide or yeast saccharomyces cerevisiae.In particularly preferred embodiment, composition promotor such as GAP-promotor, operability ground and Seq.ID.No.7 or 9, or in Seq.ID.No.13 or 15 one, or one nucleotide sequence phase coupling among the Seq.ID.No.50 to 59, and in suitable expression, express.

In another embodiment preferred, composition promotor such as GAP-promotor, operability ground comprises the nucleotide sequence coupling mutually of Profibrinolysin funtion part at least with encoding.In an especially preferred embodiment, composition promotor such as GAP-promotor, operability ground and Seq.ID.No.13,15,7 and 9, or a phase coupling among the Seq.ID.No.50 to 59, or with the nucleotide sequence coupling mutually of Seq.ID.No.11, and in suitable expression, express.

The L-glutamic acid Profibrinolysin (is used EditSeq ^TM(DNASTAR) program data calculated)

Molecular weight: 88431.67Dalton (way is paused)

791 amino acid

During pH=7.0,7.121 iso-electric points are 1.351

Glycosylation site: 0-268, N-308,0-365

(according to the serial number of the preceding Profibrinolysin of forming by 810 amino acid)

The Methionin Profibrinolysin (is used EditSeq ^TM(DNASTAR) program data calculated)

Molecular weight is: 79655.71Dalton

741 amino acid

During pH=7.0,7.492 iso-electric points are 5.287

Glycosylation site: 0-268, N-308,0-365

Miniplasminogen (is used EditSeq ^TM(DNASTAR) program data calculated)

Molecular weight is: 38169.63Dalton

348 amino acid

During pH=7.0,7.203 iso-electric points are 0.893

Glycosylation site: do not have

Microplasminogen (is used EditSeq ^TM(DNASTAR) program data calculated)

Molecular weight is: 27230.41Dalton

249 amino acid

During pH=7.0,7.934 iso-electric points are 3.733

Glycosylation site: do not have

Method of the present invention is described in detail as follows:

The fusion product that step a) of the present invention produces can be integrated in the expression vector that is suitable for microorganism.Described expression vector is preferably selected from following carrier: pPICZ α A, B and C, pPICZA, B and C, pGAPZ α A, B and C, pGAPZA, B and C, pPIC6 α A, B and C, pPIC6A, B and C, and pAO815, pPIC3.5K and pPIC9K.More preferably use the method that connects, import expression vector.Before connecting with the T4DNA ligase enzyme, PCR product and expression vector preferably use restriction endonuclease KspI and XhoI enzyme to cut.Nucleic acid after the connection can be transformed in the preferred colibacillary microorganism by electroporation, can extract DNA from thus obtained microorganism, and preferably use restriction endonuclease XhoI or SfuI and KspI, carry out the enzyme cutting from.Thus obtained nucleic acid can be a kind of plasmid, is preferably selected from following plasmid: pMHS476.1, pSM54.2, pSM49.8, pSM82.1, und pSM58.1, pAC37.1, pJW9.1, pPLG1.1, pPLG2.1, pPLG3.2, pPLG4.2, pPLG5.3, pPLG6.1, pPLG7.1, pPLG8.3, pPLG9.1, pPLG10.1, pPLG11.2, pPLG11.2, pPLG13.1, pPLG14.2, pPLG15.1, pPLG16.3, pPLG17.2, pPLG18.1, pPLG19.2 or pPLG20.1.Two few nucleic acid primers that are used for above-mentioned amplification can be preferably selected from following sequence: NO34 (Seq.ID No.1), NO36 (Seq.ID No.2), NO36a (Seq.ID No.19), NO36b (Seq.ID No.20), NO36c (Seq.ID No.21), NO36d (Seq.ID No.22), NO36e (Seq.ID No.23), NO36f (Seq.ID No.24), NO36g (Seq.ID No.25), NO36h (Seq.ID No.26), NO36i (Seq.ID No.27), NO36j (Seq.IDNo.28), NO57 (Seq.ID No.3), NO37 (Seq.ID No.4), NO35 (Seq.ID No.5) or NO56 (Seq.ID No.6).

According to the present invention, following embodiment is particularly preferred:

The codon of-proteins encoded enzyme Kex2 restriction enzyme site and Profibrinolysin fusion gene, its distinctive nucleotide sequence is shown in the sequence of Seq.ID No.7 or 13.

The codon of-proteins encoded enzyme Kex2 restriction enzyme site and Profibrinolysin fusion rotein, its distinctive aminoacid sequence feature shows in the sequence of Seq.ID No.8 or 14.

The codon of-proteins encoded enzyme Kex2, Ste13 restriction enzyme site and Profibrinolysin fusion gene, its distinctive nucleotide sequence is shown in the sequence of Seq.ID No.9 or 15.

The codon of-proteins encoded enzyme Kex2, Ste13 restriction enzyme site and Profibrinolysin fusion gene, its distinctive aminoacid sequence is shown in the sequence of Seq.ID No.10 or 16.

Preferably the above-mentioned plasmid that will optimize from above-mentioned a plurality of plasmids is transformed in the host microorganism.Can adopt method for transformation as electroporation.The microorganism of using is preferably eukaryotic microorganisms, and it is the branch of fungi.Preferred microorganism is an Ascomycota, wherein preferred Sacchariomycotina, more preferably yeast guiding principle wherein, Saccharomycetales more preferably, more preferably Saccharomycetaceae section wherein particularly preferably is Pichia, yeast belong, the inferior genus of the Chinese (Hansenula) and Aspergillus (Aspergillus).

The particularly preferred embodiment according to the present invention, in the host microorganism that has transformed the fusion product that step a) produced, encode at least that the nucleotide sequence of Profibrinolysin funtion part is the overexpression state, and secrete described Profibrinolysin funtion part at least, preferably be secreted in the substratum.According to another embodiment preferred, the nucleotide sequence of Profibrinolysin funtion part is among the sequence Seq.IDNo.60,61,62,63,64,65 or 66.According to another kind of embodiment preferred, the nucleotide sequence of Profibrinolysin funtion part is consistent with complete Profibrinolysin sequence.Utilize recombinant products production method of the present invention, preferably produce the functional Profibrinolysin of people.

Profibrinolysin according to recombinant products production method acquisition of the present invention, the plasmin that perhaps described Profibrinolysin obtains after the proteolytic enzyme effect, all can be used in the medicine of production for treating wound, be particularly useful for the slow or difficult more sexual trauma of treatment healing, also can be used in treatment thrombosis incident or prevention thrombosis incident.

Find that in addition Profibrinolysin produced according to the invention and the plasmin that therefrom obtains have the anti-freezing characteristic.These advantageous feature make Profibrinolysin and/or plasmin, can also use as antithrombotic agent and anticoagulating active agent, can be used for preventing and/or treating following disease: heart attack, thrombosis, heart valve postoperative restenosis, anoxic, ischemic, coagulation necrosis, vasculitis; The successive treatment that can be used for following disease: heart attack, bypass surgery, revascularization, balloon dilatation.Profibrinolysin also can be used for inaccessible perfusion more coronarius in leading to again of the intraictal thromboembolism treatment of acute heart disease, arteriovenous shunt and the acute cardiac onste.Profibrinolysin produced according to the invention also can be used for prevention and treats following disease: acute pulmonary embolism, phlebothrombosis New Development or old is condensed, acute and subacute arterial thrombus, phlebothrombosis, acute acra obstruction of artery, the chronic occlusion arteriopathy, arteriovenous shunt place thrombosis, buttocks and acra deep venous thrombosis, the early stage thrombosis of inaccessible (desobliterated) angiosomes, acute eye central vessel embolism, I type Profibrinolysin lacks the conjunctivitis that causes, burn and frostbite, disseminated intravascular coagulation when alkali or acid burn and shock.

When suffering from above-mentioned disease, preferably use Profibrinolysin and/or plasmin with antithrombotics.The antithrombotics that is suitable for is heparin, heparin derivatives or acetylsalicylic acid.

Therefore, pharmaceutical composition of the present invention is also very important, this pharmaceutical composition comprises: the Profibrinolysin or the thus obtained plasmin that obtain according to recombinant products production method of the present invention, and acceptable matrix, additive and/or solvent on the required pharmacology.In addition, preferred pharmaceutical compositions can comprise the anticoagulating active agent, especially heparin, heparin derivatives or acetylsalicylic acid.

Profibrinolysin produced according to the invention and/or thus obtained plasmin are used for the external treatment of wound, preferably are used for the topical therapeutic of wound with pharmaceutical compositions.In this used, the working concentration of Profibrinolysin and/or plasmin was to contain Profibrinolysin and/or the plasmin of 0.01-500U in every gram pharmaceutical composition, be preferably 0.1-500U, more preferably 0.1-250U further is preferably 0.5-250U, particularly preferably is 1-150U.If replace material with plaster or other dressing, need consider every 2cm respectively as semi-solid preparations such as ointment, paste or jellies ²Above-mentioned given the usefulness concentration and the dressing surface-area of plaster surface-area.

According to the required type used of currently known methods and suitable dosage, use solid-state, liquid matrix commonly used or thinner and the pharmaceutical adjuvant that generally uses is produced pharmaceutical composition of the present invention.Preferred drug substances or composition are for being fit to the pharmaceutical dosage form of local topical.Described pharmaceutical dosage form for example, ointment, paste, jelly, dressing, powder, emulsion, suspension agent or other special preparation attitudes, this special preparation such as nano-dispersed system, comprise liposome, nano-emulsion or lipid nanometer particulate form, also comprise preparation, the polymer emulsion stable or that particulate is stable of surfactant-free.

The production method of the known various preparations of those of ordinary skills and different application methodes, and as " Remington ' s Pharmaceutical Sciences, Mack Publishing Co., Easton PA " in have a detailed description.

Under following situation, it is suitable that pharmaceutical composition is made for the form that is used for parenteral admin: pharmaceutical composition is used to prevent and/or treat following disease, restenosis, anoxic, ischemic, coagulation necrosis, vasculitis and acute cardiac onste behind heart attack, thrombosis, the cardiac valve procedure; With the successive treatment that is used for following disease: heart attack, bypass surgery, revascularization and balloon dilatation.

In addition, described pharmaceutical composition is the application of suitable various systems also, comprise acute pulmonary embolism, thromboembolism treatment in the acute cardiac onste, phlebothrombosis New Development or old, acute and subacute arterial thrombus, arteriovenous shunt is logical again, venous thrombosis, inaccessible perfusion more coronarius in the acute cardiac onste, acute acra obstruction of artery, the chronic occlusion arteriopathy, the arteriovenous shunt thrombus, buttocks and acra deep venous thrombosis, the early stage thrombosis in occluding vascular zone, acute eye central vessel embolism, I type Profibrinolysin lacks the conjunctivitis that causes, burn, disseminated intravascular coagulation when alkali or acid burn and frostbite and shock.

Described pharmaceutical composition also can be used for the situation that Profibrinolysin lacks, for example heredity or congenital (congenital) Profibrinolysin lack (homozygote I type Profibrinolysin shortage), and described Profibrinolysin lacks can cause ligneous conjunctivitis (conjunctivitis lignosa) or thrombophilia state.Also can treat above-mentioned disease by for example intravenous injection recombinant plasminogen, described Profibrinolysin comprises Glu-, Lys-, little-and little-Profibrinolysin and derivative thereof (Heinz etc., Klin.Monatsblatt Augenheilkunde 2002,219 (3): 156-8).

Another kind of possible application is, can eliminate the respiratory tract pseudomembrane by using Profibrinolysin, makes respiratory tract normalizing, and makes the faster healing of wound.Described application be used for the newborn infant (The NewEngland Journal of Medicine 1998,339,23,1679-1686).

The Profibrinolysin that reorganization produces uses with thus obtained plasmin, or in pharmaceutical composition, only add plasmin, all be applicable to prevent and/or treat following disease: acute pulmonary embolism, phlebothrombosis New Development or old, acute and subacute arteriovenous thrombus, arteriovenous shunt is logical again, venous thrombosis, inaccessible perfusion more coronarius in the acute cardiac onste, acute acra obstruction of artery, the chronic occlusion arteriopathy, the arteriovenous shunt thrombosis, buttocks and acra venous thrombosis, the early stage thrombosis in occluding vascular zone, acute eye central vessel embolism, I type Profibrinolysin lacks the conjunctivitis that causes, burn, disseminated intravascular coagulation when alkali or acid burn and frostbite and shock.

The pharmaceutical composition that preferably contains the Profibrinolysin of recombinant production of the present invention, it can be suitable for the topical therapeutic of following disease: burn, frostbite, alkali or acid burn, damage and/or wound, especially difficult more sexual trauma.Preferred described recombinant plasminogen uses with at least a activator, and described plasminogen activator can be for example urokinase or streptokinase.Another kind of preferable methods is, before using the Profibrinolysin that the present invention produces, by activator Profibrinolysin is activated wholly or in part and to be plasmin, and use it for above-mentioned indication, the pharmaceutical preparation of this method comprises, single form that adds Profibrinolysin with plasmin or plasmin.

Considerable especially parenteral admin approach is that intravenously is used, intra-arterial is used, intraperitoneal is used, subcutaneous administration and muscle are used.If adopt the parenteral admin approach, protein concn scope in the solution of injection or transfusion usefulness is at 0.1-100 1,000,000 units/10ml solution, be preferably 10-100 1,000,000 units/10ml, more preferably 1-10 1,000,000 units/10ml is preferably 3-5 1,000,000 units/10ml especially.If the employing oral way, then protein concn is 0.1-100, and 000 units/gram preparation is preferably 100-80, and 000 units/gram is preferably 1 especially, 000-50,000 units/gram.

Pharmaceutical preparation with further advantage for example, contains plaster, dressings or other outer application materials of proteolytic enzyme.These preparations are particularly suitable for the local application of wound healing, or are used for following treatment of diseases: burn, frostbite, alkali or acid burn and/or damage.The Profibrinolysin of preferred recombinant production of the present invention is used in the pharmaceutical composition, described pharmaceutical composition comprises that especially Wound healing agent, ointment and other are used for the material of external application, they and at least a activator (described plasminogen activator such as urokinase, streptokinase), or activate the plasmin that forms in advance by aforementioned activator and use together, in described pharmaceutical composition and preparation and/or on, plasmin uses with Profibrinolysin potentially, or uses with at least a activator potentially.In plaster and outer application material and/or on especially preferably use Profibrinolysin, preferably Profibrinolysin and a kind of activator; Or plasmin or plasmin add Profibrinolysin and a kind of activator, and described preparation is applicable to trauma care, is particularly useful for the difficult more treatment of sexual trauma, also can be used for treating burn, frostbite, alkali or acid burn, or other damages.

Dressing, wound healing dressing or plaster, contain Profibrinolysin and/or thus obtained plasmin that the present invention produces, wherein, every square centimeter of pharmaceutical preparation contains 0.01-500U Profibrinolysin and/or plasmin, and preferred every square centimeter of dressing or plaster contain 0.1-500U Profibrinolysin and/or plasmin respectively.In the pharmaceutical preparation in preferred every square centimeter of plaster or the dressing, the Profibrinolysin that contains and/or the concentration of consequent plasmin are 0.1-250U, and further 0.5-250U more preferably is preferably 1-150U especially.

For activating the 1mg Profibrinolysin, the consumption of urokinase is 1ng (nanogram)-100 μ g (microgram), and preferable amount is 10ng-10 μ g.For activating the 1mg Profibrinolysin, the consumption of streptokinase is 1 μ g-1mg, and preferable amount is 3 μ g-300 μ g.Be activating the 1mg Profibrinolysin, is 10ng-100 μ g from the consumption of the proteolytic enzyme in streptomyces griseus (S.griseus) source, and preferable amount is 100ng-10 μ g.For activating the 1mg Profibrinolysin, the consumption of proteolytic enzyme VIII is 10ng-100 μ g, and preferable amount is 100ng-10 μ g.

The nucleotide sequence of coding Profibrinolysin funtion part is dna sequence dna preferably.

In addition, plasmid related to the present invention is as follows:

Plasmid pPLG1.1

Plasmid pPLG2.1

Plasmid pPLG3.2

Plasmid pPLG4.2

Plasmid pPLG5.3

Plasmid pPLG6.1

Plasmid pPLG7.1

Plasmid pPLG8.3

Plasmid pPLG9.1

Plasmid pPLG10.1

Plasmid pPLG11.2

Plasmid pPLG13.1

Plasmid pPLG14.2

Plasmid pPLG15.1

Plasmid pPLG16.3

Plasmid pPLG17.2

Plasmid pPLG18.1

Plasmid pPLG19.2

Plasmid pPLG20.1

Plasmid pMHS476.1 (preserving number: DSM 14678)

Plasmid pSM54.2 (preserving number: DSM 14682)

Plasmid pSM49.8 (preserving number: DSM 14681)

Plasmid pSM82.1 (preserving number: DSM 14679)

Plasmid pSM58.1 (preserving number: DSM 14680)

Plasmid pAC37.1 (preserving number: DSM 15369)

Plasmid pJW9.1 (preserving number: DSM 15368).

(preserving number refers to the deposit number (GermanCollection of Microorganisms and Cell Cultures Ltd., MascheroderWeg 1b, D-38124 Braunschweig) at German microorganism and cell culture preservation center.

In addition, the present invention relates to be fit to the dna sequence dna of expression, this sequence comprises the nucleotide sequence of the Profibrinolysin funtion part of encoding at least, and this nucleotide sequence can obtain according to recombinant method for production of the present invention.In addition, the invention still further relates to host microorganism, one section nucleotide sequence that it comprises fusion product and derives thus, described fusion product is seen above-mentioned steps a).The invention still further relates to carrier, dna molecular or RNA molecule, the nucleotide sequence that described carrier, dna molecular or RNA molecule comprise fusion product and derive thus, described fusion product is seen above-mentioned steps a).

At last, the invention still further relates to the screening method of identifying plasminogen activator, described activator especially refers to Profibrinolysin activity proteolytic enzyme, wherein, uses the functional Profibrinolysin of producing according to the production method of above-mentioned recombinant products.According to this purpose, the plasmin that obtains after the preferred protease preincubation is measured activity with the functional Profibrinolysin of producing according to the method for the invention.Can use one section synthetic peptide to measure the activity of gained plasmin.Especially preferably use N-tosyl (tolylsulfonyl)-Gly-Pro-Lys-pNA to measure the activity of gained plasmin.

Description of drawings

Use accompanying drawing to explain the present invention in more detail, accompanying drawing is as follows:

Fig. 1: the physical map of plasmid pMHS476.1 (5682bp).Alpha factor propetide protogene is connected with people's Methionin Profibrinolysin gene by the codon of Kex2 restriction enzyme site, and described alpha factor propetide protogene is under the control of AOX1 promotor.

Fig. 2: the physical map of plasmid pSM54.2 (5694bp).Alpha factor propetide protogene is connected with people's Methionin Profibrinolysin gene by the codon of a Kex2 and two Ste13 restriction enzyme sites, and described alpha factor propetide protogene is under the control of AOX1 promotor.

Fig. 3: the physical map of plasmid pSM49.8 (5715bp).The Profibrinolysin gene is under the control of AOX1 promotor before the people.

Fig. 4: the physical map of plasmid pSM82.1 (5913bp).Alpha factor propetide protogene is connected with people's Methionin Profibrinolysin gene by the codon of Kex2 restriction enzyme site, and described alpha factor propetide protogene is under the control of AOX1 promotor.

Fig. 5: the physical map of plasmid pSM58.1 (5925bp).Alpha factor propetide protogene is connected with human glutamic acid Profibrinolysin gene by the codon of a Kex2 and two Ste13 restriction enzyme sites, and described alpha factor propetide protogene is under the control of AOX1 promotor.

Fig. 6: the physical map of plasmid pAC37.1 (11400bp).Alpha factor propetide protogene is connected with people's Methionin Profibrinolysin gene by the codon of a Kex2 and two Ste13 restriction enzyme sites, and described alpha factor propetide protogene is under the control of AOX1 promotor.

Fig. 7: the physical map of plasmid pJW9.1 (5925bp).Alpha factor propetide protogene is connected with people's Methionin Profibrinolysin gene by the codon of a Kex2 and two Ste13 restriction enzyme sites, and described alpha factor propetide protogene is under the control of GAP promotor.

Fig. 8: the physical map of plasmid pPLG1.1.Alpha factor propetide protogene is connected with people's Miniplasminogen gene by the codon of a Kex2 and two Ste13 restriction enzyme sites, and described alpha factor propetide protogene is under the control of AOX1 promotor.

Fig. 9: the physical map of plasmid pPLG11.2.Alpha factor propetide protogene is connected with people's Miniplasminogen gene by the codon of a Kex2 and two Ste13 restriction enzyme sites, and described alpha factor propetide protogene is under the control of GAP promotor.

Figure 10: by Kl  rhof analyzing and testing fibrinolytic activity.

Figure 11: the cut of purifying is in the electrophoretic result of 7.5% SDS-PAGE among the embodiment 6g.

Embodiment

According to the present invention, all microorganisms that can have glycosylation and secretory protein (if desired) characteristic can be as host organisms.Be exemplified below: the filamentous fungus of yeast saccharomyces cerevisiae, pichia pastoris phaff, pichia methanolica, multiple-shaped nuohan inferior yeast Aspergillus.

Special functional Profibrinolysin and the plasmin of considering that use is produced respectively according to the present invention in pharmaceutical preparation.In such preparation, functional Profibrinolysin can mix use with the acceptable matrix of medicine, assistant agent and other assistant agents well known by persons skilled in the art or additive.

Exist the Kex2 restriction enzyme site that can be cut by Kex2 proteolytic enzyme on the former peptide, described Kex2 proteolytic enzyme is arranged in golgi body.This proteolytic enzyme also is known as proteolytic enzyme YscF or Kexin, is a kind ofly can process former proteic serine protease, and it cuts C-terminal from primary amino acid to (basic amino acidpairs) (as Lys-Arg).

Exist the Ste13 restriction enzyme site that can be cut by Ste13 proteolytic enzyme on the former peptide, described Ste13 proteolytic enzyme is arranged in golgi body.Proteolytic enzyme Ste13 (also being known as proteolytic enzyme YscVI or dipeptide aminopeptidase A) is distributed in the golgi body in late period, and the Xaa-Ala dipeptides of hydrolyzing N end progressively is as the Xaa-Ala dipeptides of the N end of the immature alpha factor of yeast saccharomyces cerevisiae.

Except the restriction enzyme site of Proteinase K ex2 and Ste13, also can insert other restriction enzyme sites, the proteolytic enzyme that described other restriction enzyme sites can be positioned in endoplasmic reticulum or the golgi body is identified as substrate.

Also can be only merge with the signal sequence (propetide) of Profibrinolysin gene, this signal sequence is responsible for product is transported into endoplasmic reticulum, that is, in described process such as the former peptide of the hybridization factor of yeast saccharomyces cerevisiae and nonessential.

Those skilled in the art know the method for the microbiology, molecular biology and the protein chemistry that relate in an embodiment.Below be bibliography: Maniatis etc., and Molecular Cloning:A Laboratory Manual (molecular cloning: laboratory manual), Cold Spring Harbor press (cold spring port press), 1989 (10); Gassen ﹠amp; Schrimpf, Genetic EngineeringMethods (gene engineering method), Spektrum Akademischer Verlag, Heidelberg, 1999 (11); EasySelect ^TMPichia Expression Kit Instruction Manual (EasySelect ^TMPichia expresses the test kit operational manual), Invitrogen, Groningen, Holland, catalog number (Cat.No.): K1740-01.Pichia pastoris phaff bacterial strain and expression system all can be available from Invitrogen, and describe to some extent in the aforesaid operations handbook.

PPICZA, B and C are the pichia pastoris phaff expression vectors of 3.3kb, and this carrier is shorter.There is zeorin (zeocin) resistant gene on the carrier, is used for directly screening the pichia pastoris phaff transformant.In addition, there is C end flag sequence on this carrier, is used for fast purifying and detect fusion rotein.PPICZalpha A, B and C are the pichia pastoris phaff expression vectors of 3.6kb, also have zeorin resistant gene and above-mentioned C end flag sequence.In addition, they also comprise yeast saccharomyces cerevisiae alpha factor secretion signal, are used for albumen is secreted into substratum effectively.

In addition, Profibrinolysin can be activated.Can be activated after Profibrinolysin and a kind of proteolytic enzyme are hatched, described proteolytic enzyme adopts screening method of the present invention to identify.

Preferred Profibrinolysin is with following enzyme incubation: the proteolytic enzyme, proteolytic enzyme VIII, proteolytic enzyme XVIII, ficin, Zinc metalloproteinase, clostripain, L-glutamic acid-C end endoproteinase, proteolytic enzyme XIII, protease A, trypsinase, aspartic acid-N end endoproteinase or the elastoser that derive from streptomyces griseus.

Further it is contemplated that, by t-PA, u-PA or vb-PA[vampire-PA] hatch plasminogen activation with Profibrinolysin.

In another kind of embodiment preferred, Profibrinolysin is activated by hatching with staphylokinase and streptokinase.Streptokinase or staphylokinase and Profibrinolysin form a kind of 1: 1 mixture.By forming this mixture, the conformation of the Profibrinolysin in the mixture changes, thereby it has proteolytic activity, and can make it to change into plasmin by plasminogen activation.

According to the functional Profibrinolysin of this recombination method acquisition, or the functional Profibrinolysin of activatory, the ability of hydrolysis of fibrin all had.Described Profibrinolysin also has the ability that activates former matrix metalloprotease and somatomedin.

To illustrate in greater detail the present invention by the following example.

Embodiment 1a: amplification Methionin Profibrinolysin gene, described gene inserts the codon of Kex2 restriction enzyme site at 5 ' end

Use extraction reagent kit in the qiagen plasmid (QIAGEN, Hilden), extraction plasmid pPLGKG from HB101 e. coli strains (containing pPLGKG) (Forsgren etc., FEBS Lett.1987 March 23; 213 (2): 254-60 (2)), before this plasmid contains-L-glutamic acid-Profibrinolysin (pre-Glu-plasminogen) gene.(Roche, Mannheim) enzyme is cut 150ng pPLGKG-DNA and is made it linearizing, uses QIAquick PCR purification kit (QIAGEN, Hilden) purify DNA subsequently with the EcoRI restriction enzyme of 10U.With primer NO34 (Seq.ID No.1) and NO36 (Seq.ID No.2) amplification Profibrinolysin gene.Except that with the base of Profibrinolysin gene complementation, Oligonucleolide primers NO36 also has the codon of Kex2 restriction enzyme site.Each reaction system comprises 0.5U Pwo-DNA-polysaccharase, and (Hybaid, Heidelberg), Oligonucleolide primers, 200 μ M dNTP, the linear pPLGKG-DNA of 3ng and the reaction buffer separately of 400nM, final volume is 50 μ l.Primer is 58 ℃ in conjunction with temperature.

Whether detect PCR product band expection size by agargel electrophoresis correct, and with QIAquick PCR purification kit purifying.

Embodiment 1b: the Profibrinolysin gene clone in the pPICZaA carrier

Get KspI and XhoI (Roche, the mannheim) digestion with restriction enzyme of the PCR product of 400ng with each 10U.Plasmid pPICZ α AA (Invitrogen, Groningen, Holland) contains the former sequence of the propetide that derives from yeast saccharomyces cerevisiae α-factor, and the DNA that gets the described plasmid of 300ng also uses KspI and the XhoI digestion with restriction enzyme of 10U.At the DNA that separates on 0.9% sepharose after enzyme is cut, (QIAGEN Hilden) reclaims the fragment that obtains from gel with the QIAquick gel extraction kit.Carrier DNA and the mixed 1U T4-DNA-ligase enzyme (Roche, mannheim) that is incorporated in of insertion DNA are acted on down, and 4 ℃ of connections are spent the night.

Use QIAquick PCR purification kit subsequently, this batch of purifying connects product D NA, and described purified product is used for transformed into escherichia coli JM109 by electroporation.

With the JM109 Bacillus coli cells after the electroporation conversion, inoculation is gone in the 1ml SOC substratum, cultivates 1 hour for 37 ℃, be laid on then and be added with 20 μ g/ μ l zeorin (Invitrogen, Groningen, Holland) on the LB agar solid agar base, 37 ℃ are incubated overnight.

From thus obtained intestinal bacteria, (QIAGEN plasmid mini kit, QIAGEN Hilden) extract DNA to use the little extraction reagent kit of qiagen plasmid, behind XhoI and KspI digestion with restriction enzyme, isolate 300ng nucleic acid by agarose gel electrophoresis.Isolated plasmid comprises the fragment of expection size, and this plasmid is known as pMHS476.1 (Fig. 1).By sequential analysis, determine that the sequence of fusion gene is correct, this fusion gene is formed (Seq.ID No.7) by the codon that is derived from yeast saccharomyces cerevisiae α-factor propetide protogene, Methionin Profibrinolysin gene and Proteinase K ex2 restriction enzyme site.

Embodiment 1c: transform pichia pastoris phaff with plasmid pMHS476.1

(QIAGEN plasmid midi kit, QIAGEN Hilden) from JM109 e. coli strains (containing pMHS476.1), extract the expression vector pMHS476.1 plasmid DNA of Profibrinolysin to use extraction reagent kit in the qiagen plasmid.(New England Biolabs, Frankfurt) enzyme is cut 10 μ g pMHS476.1-DNA and is made its linearizing, according to EasySelect with 100U PmeI ^TMPichia expresses method shown in the test kit operational manual, its electroporation is transformed in the pichia pastoris phaff KM71H strain, the genotype of this KM71H strain is his4::HIS 4 arg 4 aoxl:: the ARG 4 of pichia pastoris phaff Y-11430 strain (Northern Regional Research Laboratories, Peoria, the U.S.).Describedly be cloned in the YPDS solid medium growth after three to four days, be plated on the YPDS solid medium that contains 100 μ g/ml zeorins and cultivate, and be used to inoculate liquid substratum.With described clone's called after pichia pastoris phaff KM71H/pMHS476.1-1/a, " a " representative is since 1 clone's serial number herein.

Embodiment 1d: growth and the inducing of Profibrinolysin genetic expression of pichia pastoris phaff KM71H/pMHS476.1-1/1 to-1/3

For preparing pre-culture (precultures), 100ml BMGY substratum (EasySelect ^TMPichia expresses the test kit operation instructions) place the Schikane flask of 1L, under 28 ℃ and 250rpm (commentaries on classics/per minute) condition, cultivate, to OD ₆₀₀Reach 20-30.Then, pre-culture with 4645g (centrifugal force), was descended centrifugal 10 minutes at 4 ℃.For the wet mass concentration that makes thalline reaches 80g/L, the bacterium of collecting is resuspended in the BMMY-substratum (containing 0.5% methyl alcohol).Get the described main culture of 60ml and cultivated 118 hours in the Schikane of 300ml flask, condition is 28 ℃ and 250rpm.Add 2% methyl alcohol after 24 and 72 hours.The Schikane flask must be with the high speed rocking-turn of 250rpm, provide competent oxygen to give bacterium liquid, necessary condition when this is to use the AOX promotor.

Embodiment 1e: after using streptokinase to activate, in main culture supernatants, detect the activity of Profibrinolysin

The sample of main culture under the condition of 16000g centrifugal 10 minutes.Get 300 μ l supernatant liquors, (Sigma Deisenhofen), cultivates 20min under 37 ℃ of conditions to add 1 μ l streptokinase (S8026).Sodium phosphate buffer (pH=8), 0.36mM CaCl at the 100mM of 750 μ l (every liter of mmole) ₂, among the 0.9%NaCl, (9.5mg is dissolved in 75mg glycine/10ml, 2% tween (Tween to add 100 μ l N-tosyl-Gly-Pro-Lys-pNA solution ^) 20), 37 ℃ of incubations 10 minutes.Add 250 μ l and start reaction, at 37 ℃ of further incubations with the pretreated supernatant liquor of streptokinase.Measure light absorption value at the 405nm place, show that consumption is increasing.Also measure the supernatant liquor of parallel group of pichia pastoris phaff KM71H culture in contrast simultaneously and do not use streptokinase activated supernatant liquor.For example, after sample was induced through 72 hours, detecting its activity value (1U/l is defined as and transforms 1 μ mol N-tosyl-Gly-Pro-Lys-pNA in 1 liter of culture supernatants 1 minute) is, KM71H/pMHS476.1-1/1:2U/l, KM71H/pMHS476.1-1/2:2U/l, KM71H/pMHS476.1-1/3:1U/l.Through after inducing 118 hours, detect its activity value and be KM71H/pMHS476.1-1/1:7U/l, KM71H/pMHS476.1-1/2:9U/l, KM71H/pMHS476.1-1/3:8U/l.

Embodiment 2a: amplification Methionin Profibrinolysin gene, described gene 5 ' end inserts the codon of a Kex2 and two Ste13 restriction enzyme sites

With two Oligonucleolide primers NO34 and NO57 (Seq.ID No.3) the Methionin Profibrinolysin gene that increases, described gene inserts the codon of a Kex2 of coding and two Ste13 restriction enzyme sites, this amplified production is used to insert carrier pPICZ α A, and amplification condition is as described in the embodiment 1a.Oligonucleolide primers NO57 the Profibrinolysin gene complementary base by, have the codon of Kex2 and Ste13 restriction enzyme site.

Embodiment 2b: the Methionin Profibrinolysin gene that increases among the clone embodiment 2a is in pPICZ α A carrier

Utilize and the similar method of embodiment 1b, with the gene clone of people's Methionin Profibrinolysin in the carrier pPICZ α A producing fusion gene, this fusion gene is by the propetide protogene of yeast saccharomyces cerevisiae alpha factor and inserted Proteinase K ex2 and the human plasminogen genomic constitution of Ste13 restriction enzyme site codon.The plasmid called after pSM54.2 (Fig. 2) that obtains.Through sequential analysis, confirm that sequence (Seq.ID No.9) is correct.

Embodiment 2c: transform pichia pastoris phaff with plasmid pSM54.2

The method of conversion pMHS476.1 described in embodiment 1c transforms pichia pastoris phaff KM71H strain with plasmid pSM54.2.The clone's called after pichia pastoris phaff KM71H/pSM54.2-1/a that obtains, " a " still represents the serial number since 1 clone herein.

Embodiment 2d: cultivation and the inducing of Profibrinolysin gene of pichia pastoris phaff KM71H/pSM54.2-1/1 to-1/3

The production of pre-culture and main culture, and with the method for methanol induction, all with embodiment 1d in similar.

Embodiment 2e: after using streptokinase to activate, the active mensuration of Profibrinolysin in the sample of main culture

The determination of activity of the Profibrinolysin after streptokinase activates is seen among the embodiment 1e for extremely-1/3 description of KM71H/pMHS476.1-1/1.After inducing 72 hours, the activity value of sample is as follows: KM71H/pSM54.2-1/1:2U/l, KM71H/pSM54.2-1/2:8U/l, KM71H/pSM54.2-1/3:6U/l.Induce the activity value that records after 118 hours as follows: KM71H/pSM54.2-1/1:8U/l, KM71H/pSM54.2-1/2:17U/l, KM71H/pSM54.2-1/3:13U/l.

Embodiment 3a: amplification has the Profibrinolysin gene of native signal sequence, and is cloned in the pPICZA carrier; Transform pichia pastoris phaff

With two Oligonucleolide primers NO34 and NO37 (Seq.ID No.4) the Profibrinolysin gene that increases, this gene has the sequence of code book body signal peptide (preceding Profibrinolysin), and amplified production is inserted carrier pPICZA, and employed condition is as described in the embodiment 1a.

With the method described in the similar embodiment 1b, carrier and PCR product are all used SfuI and KspI digestion with restriction enzyme, then the Profibrinolysin gene clone in the pPICZA carrier.The plasmid called after pSM49.8 (Fig. 3) that obtains.Through sequential analysis, confirm that sequence (Seq.ID No.11) is correct.

To the description of pMHS476.1, transform pichia pastoris phaff KM71H strain among the similar embodiment 1c with plasmid pSM49.8.The clone's called after pichia pastoris phaff KM71H/pSM49.8-1/a that obtains, " a " still represents the serial number since 1 clone herein.

Embodiment 4a: amplification human glutamic acid Profibrinolysin gene, and be cloned among the expression vector pPICZ α A, described human glutamic acid Profibrinolysin gene has inserted the codon of Kex2 restriction enzyme site; Transform pichia pastoris phaff

Use two Oligonucleolide primers NO34 and NO35 (Seq.ID No.5), amplification L-glutamic acid Profibrinolysin gene, and be cloned among the carrier pPICZ α A, described L-glutamic acid Profibrinolysin gene has inserted the codon of Kex2 restriction enzyme site, and the used condition of described process is as described in the embodiment 1a.Oligonucleolide primers NO35 is at the other codon with Kex2 restriction enzyme site of the complementary sequence of L-glutamic acid Profibrinolysin gene.

Is in order to produce fusion gene with the gene clone of L-glutamic acid Profibrinolysin in carrier pPICZ α A, described fusion gene is by the propetide protogene of yeast saccharomyces cerevisiae alpha factor, inserted the human glutamic acid Profibrinolysin genomic constitution of Proteinase K ex2 restriction enzyme site codon, described process is similar to the described clone's process of embodiment 1b.

The plasmid called after pSM82.1 (Fig. 4) that obtains.Through sequential analysis, confirm that sequence (Seq.IDNo.13) is correct.

To the description of pMHS476.1, transform pichia pastoris phaff KM71H strain among the similar embodiment 1c with plasmid pSM82.1.The clone's called after pichia pastoris phaff KM71H/pSM82.1/a that obtains, " a " still represents the serial number since 1 clone herein.

Embodiment 5a: the gene of amplification human glutamic acid Profibrinolysin also is cloned into expression vector pPICZa A, and the gene of described human glutamic acid Profibrinolysin inserts the codon of encoded K ex2 and two Ste13 restriction enzyme sites at 5 ' end; Transform pichia pastoris phaff

Utilize two Oligonucleolide primers NO34 and NO56 (Seq.ID No.6), the gene of amplification L-glutamic acid Profibrinolysin, and be cloned among the expression vector pPICZa A, the gene of described L-glutamic acid Profibrinolysin has inserted the codon of encoded K ex2 and two Ste13 restriction enzyme sites, and concrete amplification condition is seen embodiment 1a.Primer NO56 is at the other codon with Kex2 and Ste13 restriction enzyme site of the complementary base of L-glutamic acid Profibrinolysin gene.

With the gene clone of human glutamic acid Profibrinolysin in carrier pPICZa A, to produce fusion gene, this fusion gene is by the propetide protogene of a factor of coding yeast saccharomyces cerevisiae, inserted the human glutamic acid Profibrinolysin genomic constitution of proteins encoded enzyme Kex2 and Ste13 restriction enzyme site sequence, the step of mentioning among concrete steps and the embodiment 1b is similar.The plasmid that obtains is called pSM58.1 (Fig. 5).By sequential analysis, confirm that sequence (Seq.ID No.15) is correct.

As the pMHS476.1 described in the embodiment 1c, transform pichia pastoris phaff KM71H strain with plasmid pSM58.1, the clone's called after pichia pastoris phaff KM71H/pSM58.1/a that obtains, " a " still represents the serial number since 1 clone herein.

Embodiment 6a: from plasmid pSM54.2, obtain people's Methionin Profibrinolysin gene, and be inserted among the carrier pPIC9K

With the every kind of restriction enzyme SacI of 10U and the carrier pPIC9K (Invitrogen, Groningen, Holland) of NotI (all from RocheDiagnostics, the mannheim) cutting 150ng.Cut the Profibrinolysin expression vector pSM54.2 (seeing embodiment 2b) of 300ng equally with same enzyme.With 0.9% sepharose the dna fragmentation of cutting through enzyme is carried out electrophoretic separation then.(QIAgen gel extraction kit, Qiagen Hilden) comes out bigger fragment purification with QIAgen gel-purified test kit.Mix two bigger fragments and at 4 ℃, the T4DNA ligase enzyme effect of 1U connects down spends the night.

With with similar method described in the embodiment 1b, transformed into escherichia coli DH5 α carries out purifying and evaluation to the gained plasmid, still, for the microbiotic of screening transformant, replaces zeorin with acillin.The plasmid called after pAC37.1 (Fig. 6) of Gou Jianing like this.

Embodiment 6b: transform pichia pastoris phaff with plasmid pAC37.1

As transforming the pichia pastoris phaff KM71H strain described in the embodiment 1c with pMHS476.1, transform pichia pastoris phaff KM71 with plasmid pAC37.1, oneself cuts described plasmid pAC37.1 with the SalI enzyme and makes it linearizing.Cell inoculation after the conversion (is expressed test kit (multi-copy pichiaexpression kit instruction manual) and is cultivated referring to multiple copied Pichia in the MD-agarose substratum that does not contain Histidine.Resulting clone's called after pichia pastoris phaff KM71/pAC37.1-3/a, " a " still represents the serial number since 1 clone herein.

Embodiment 6c: the inducing of the cultivation of pichia pastoris phaff KM71/pAC37.1-3/1 and Profibrinolysin gene

The production of pre-culture and main culture and the method for methanol induction are similar with the condition of description among the embodiment 1d.The inducing sustained time was above 216 hours.At first, change 2% methyl alcohol after 24 hours into, added one time 2% methyl alcohol in per 48 hours again with 0.5% methanol induction.

Embodiment 6d: after streptokinase activates, the active mensuration of Profibrinolysin in the sample of main culture

Streptokinase activates the active measuring method of back Profibrinolysin, and is consistent with the activity determination method of KM71H/pMHS476.1-1/1 to 1/3 among the embodiment 1e.Inducing the activity of the sample that takes out after 120 hours is 120U/L; After inducing 216 hours, detectable activity is 190U/L.

Embodiment 6e: after pichia pastoris phaff KM71/pAC37.1-3/1 inducing in minimum medium (BSM) activated with streptokinase, the active mensuration of Profibrinolysin in the main culture samples

After cultivating pichia pastoris phaff KM71/pAC37.1-3/1, the cells of the centrifugal gained of 80 grams are re-suspended in 100 milliliters of minimum mediums that comprise basic salt induce at BMGY complex medium (seeing embodiment 1d).Substratum-minimum medium the component that comprises basic salt is as follows:

H ₃PO ₄, 85%:26.0ml/l; CaCl ₂2H ₂O:0.6g/l; K ₂SO ₄: 18.0g/l; MgSO ₄7H ₂O:14.0g/l; KOH:4.0g/l; Glycerine: 20ml/l; Antifoam: 1.0ml/l; Trace element solution: 8.0ml/l; Yeast growth cellulose solution (0.2g/l): 8.0ml/l.

The composition of trace element solution: H ₂SO ₄: 5.0ml/l; CuSO ₄5H ₂O:6.0g/l; KI:0.08g/l; MnSO ₄H ₂O:3.0g/l; Na ₂MoO ₄: 0.2g/l; H ₃BO ₃: 0.02g/l; CoCl ₂: 0.5g/l; ZnCl ₂: 20.0g/l; FeSO ₄7H ₂O:65.0g/l.

For inducing, add 2% methyl alcohol every day.After streptokinase activated, the active measuring method of Profibrinolysin extremely-1/3 induced the active measuring method in back consistent with mensuration KM71H/pMHS476.1-1/1 among the embodiment 1e.After inducing 120 hours, the Profibrinolysin of mensuration activity is 193U/l, and the activity of measuring after 168 hours is 289U/l.

Embodiment 6f: measure in the sample of main culture in Kl  rhof fibrinolysis test, streptokinase activates the activity of back Profibrinolysin

Kl  rhof fibrinolysis test [Stack, M.S., Pizzo, S.V., and Gonzalez-Gronow, M. (1992): Effect of desialylation on thebiological properties of human plasminogen. (asialoglycoprotein is to the influence of human plasminogen biological characteristics), Biochem.J.284,81-86 (13)] preparation as follows: with 1.5 gram GTG low melting-point agaroses, in the sodium phosphate buffer that boils the pH=7.4 that is dissolved into 75 milliliters of 50mM; With 35ml fibrinogen solution (Fibrinogen of dissolving 225mg in the sodium phosphate buffer of the pH=7.4 of every 37.5ml 50mM) and 350 μ l thrombin solution (10U/ml, sodium phosphate buffer preparation with the pH=7.4 of 50mM) mixes, do not produce bubble; The mixing solutions stirring is joined in the described agarose solution, pour culture dish then into.After solidifying, on substratum, carves scleroproein the aperture of 1mm size.

In order to measure the fibrinolytic activity that streptokinase activates the back recombinant plasminogen, in each aperture, inject the following solution of 20 μ l components, and cultivated 20 hours at 37 ℃:

1:0.5mg/ml Profibrinolysin (Roche, mannheim);

2: the culture supernatant of the KM71/pAC37.1-3/1 among the embodiment 6e;

3:0.5mg/ml Profibrinolysin is activated by streptokinase;

4: by the culture supernatant of the KM71/pAC37.1-3/1 among the streptokinase activated embodiment 6e;

5:0.25mg/ml Profibrinolysin is activated by streptokinase;

6: dilute the culture supernatant of the KM71/pAC37.1-3/1 among one times the embodiment 6e, activate by streptokinase;

7:0.125mg/ml Profibrinolysin is activated by streptokinase;

8:, activate by streptokinase with the culture supernatant of KM71/pAC37.1-3/1 among the embodiment 6e with the KM71H of quadrat method acquisition.

When streptokinase activated, (100U/ μ l, Sigma Deisenhofen) added in described each solution of 40 μ l, hatch 60 minutes under 37 ℃ with 2 μ l streptokinases.

See Figure 10 by the bacterial plaque that the solution fibrin activity is produced.

Embodiment 6g: by the affinitive layer purification Profibrinolysin, this Profibrinolysin is by producing in pichia pastoris phaff KM71/pAC37.1-3/1 reorganization

The supernatant liquor of pichia pastoris phaff KM71/pAC37.1-3/1 among the 50ml embodiment 6c/6d is dialysed down in 4 ℃ in 4 liters of 50mM sodium phosphate buffers of pH=7.5.Change dialysis buffer liquid after 24 hours, continue dialysis 24 hours.Product after the dialysis filters with the filter in 0.02 μ m aperture, carries out affinity chromatography with the sodium phosphate buffer equilibrated Methionin-agarose TM 4B post (diameter 16mm, high 95mm) (Amersham Biosciences) through 50mM, pH7.5 then.Can remove the albumen of the non-specific binding on the post by the 50mM sodium phosphate buffer of pH=7.5, the sodium-chlor of 0.5M.The bonded Profibrinolysin can be used the sodium phosphate buffer of 50mM, pH7.5, the epsilon-amino caproic acid wash-out of 0.01M.The sample that obtains can carry out electrophoresis with 7.5% SDS-PAGE, then with argentation dyeing (Figure 11).On gel, the Profibrinolysin of the reorganization that contains in the cut is in same position with the human plasminogen that adds in contrast.

Figure 11 has shown that the cut of purifying among the embodiment 6g is in the electrophoretic result of 7.5% SDS-PAGE.The implication of used abbreviation representative is as follows among Figure 11:

M: albumen size criteria (being from top to bottom: 116kDa, 66kDa, 45kDa, 35kDa)

D: dialyzate

N: not in conjunction with cut

W: clean cut

F1-F5 is the cut that comprises Profibrinolysin of wash-out

Plg: Profibrinolysin (American Diagnostica, Pfungstadt)

Embodiment 6h:, estimate the influence of pH value and substrate by the fermentation of pichia pastoris phaff KM71/pAC37.1-3/1

The pichia pastoris phaff KM71/pAC37.1-3/1 of 2ml glycerine subzero treatment is inoculated in the YEP-G substratum (10 grams per liter yeast extracts, 20 grams per liter casein peptones, 20 grams per liter glycerine) of 50ml, described YEP-G substratum shakes in the bottle at the 1 liter of wide neck that does not contain Schikanes.Cultivated 9 hours with 30 ℃, 300 rev/mins conditions.Then this culture of 5ml is inoculated in the 50ml MG substratum [5 grams per liters do not contain amino acid whose yeast nitrogenous base (nitrgen base), 20 grams per liter glycerine, 2.5 milliliters/rise biotin solution (0.2g/l)], described MG substratum shakes in the bottle at the 1 liter of wide neck that does not contain Schikanes.This second pre-culture was cultivated 16 hours with 30 ℃, 300 rev/mins condition.Main culture is fermented in the multiple fermentor tank of " stirrer-pro " (DASGIP, J ü lich), and this device can allow to carry out simultaneously 4 fermentations under the different condition.Therefore, with 15 milliliters second pre-cultivate to be inoculated in the 150ml BSM substratum (see embodiment 6e).After adding the substrate of doses, under pH=6, begin to ferment, make great efforts to keep this pH.Under the different culture condition the parallel fermentation of carrying out the results are shown in Table 1:

Table 1:

Experiment	pH	Substrate	Feed speed	OD ₆₀₀	Profibrinolysin concentration
Experiment	pH	Substrate	Feed speed	OD ₆₀₀	Profibrinolysin concentration	I II III IV	6 7 6 6	Methyl alcohol methyl alcohol methyl alcohol/glycerine methyl alcohol	1 milliliter of/hour projects combo of projects combo projects combo	187 160 270 130	1.4 mg/litre 6.1 mg/litre 10.1 mg/litre 3.4 mg/litre

The peptone that in the substratum of experiment IV, has added 30 grams per liters.Before methyl alcohol begins effect, add earlier the substratum of supplying with glycerine [500 grams per liter anhydrous glycerols, 10 milliliters/rise trace element solution, 10 milliliters/rise biotin solution (seeing embodiment 6e)] and continue 4 hours with 24 milliliters/hour constant speeds.Projects combo among experiment I, II and the IV is to determine dose function by the following formula, f (x)=P1+ (P2/1+exp (P3 (t-P4))))+(P5/ (1+exp (P6 (t-P7)))), wherein P1=0, P2=0.7, P3=0.2, P4=15, P5-P6=P7=0.As can be seen from Table 1, resulting Profibrinolysin concentration is the highest under neutral pH and mixing methyl alcohol and the glycerine dosage.

Embodiment 7a: obtain Methionin Profibrinolysin gene from pAC37.1, and be inserted among the pGAPZaA

150ng carrier pGAPZ α A (Invitrogen, Groningen, Holland) is carried out enzyme with the restriction enzyme XhoI of 10U and NotI (all from Roche Diagnostics, mannheim) to be cut.Also cut simultaneously the Profibrinolysin expression vector pAC37.1 (seeing embodiment 6a) of 300ng with these two enzymes.Dna fragmentation after agarose with 0.9% is cut enzyme carries out gel electrophoresis to be separated.The long fragment of 3073 bases that obtains after Profibrinolysin gene fragment that 2715 bases (bp) that obtain after the pAC37.1 enzyme cut are long and pGAPZ α A enzyme are cut, with QIAgen gel-purified test kit purifying (Qiagen, Hilden).Mix these two fragments then and under 4 ℃, the effect of the T4DNA ligase enzyme of 1U, connect and spend the night.

Transformed into escherichia coli DH5 α, the purifying and the method among authentication method and the embodiment 1b that obtain plasmid are similar.But, replace zeorin to screen transformant with acillin.Constructed plasmid called after pJW9.1 (Fig. 7).

Embodiment 7b: transform pichia pastoris phaff with plasmid pJW9.1

As transforming the method for pichia pastoris phaff KM71H strain with pMHS476.1 among the embodiment 1c, transform pichia pastoris phaff KM71H strain with plasmid pJW9.1, before transforming earlier with restriction endonuclease BlnI with the pJW9.1 linearizing.Cell coated plate after the conversion is (EasySelect on the YPDS-agar that contains 100 mcg/ml zeorins ^TMPichia expresses the test kit service manual) and cultivate.Resulting clone's called after pichia pastoris phaff KM71H/pJW9.1-a, " a " still represents the serial number since 1 clone herein.

Embodiment 7c:, under different glycerine feed speeds, estimate the pH value by the fermentation culture of pichia pastoris phaff KM71H/pJW9.1-3

Pre-culture and the fermentation culture method in " stirrer-pro " are seen embodiment 6i, and gained the results are shown in Table 2.

Table 2:

Experiment	pH	Substrate	Feed speed	OD ₆₀₀	The concentration of plasmin
Experiment	pH	Substrate	Feed speed	OD ₆₀₀	The concentration of plasmin	I II III IV	6.5 7.0 6.5 7.0	Glycerine glycerine glycerine glycerine	1 milliliter/hour 1 milliliter/hour 0.5 milliliter/hour 0.5 milliliter/hour	220 203 142 99	18.6 mg/litre 22.2 mg/litre 10.1 mg/litre 3.8 mg/litre

Equally, best in the result that the condition of neutral pH bottom fermentation obtains, from table, also can clearly be seen that the influence that substrate dosage (feed speed) forms product.

Embodiment 7d: the fermentation of pichia pastoris phaff KM71H/pJW9.1-3

Pichia pastoris phaff KM71H/pJW9.1-3 is inoculated in the YEP-G substratum (10 grams per liter yeast extracts, 20 grams per liter casein peptones, 20 grams per liter glycerine) of 50ml, described YEP-G substratum was cultivated 9 hours with 30 ℃, 300 rev/mins conditions in 1 liter that does not contain Schikanes wide neck bottle.(5 grams per liters do not contain amino acid whose yeast nitrogenous base, 20 grams per liter glycerine, 2.5 milliliters/rise in the biotin solution (0.2g/l), and described MG substratum is in 1 liter that does not contain Schikanes wide neck bottle then the described culture of 10ml to be inoculated into 40ml MG substratum.Condition with 30 ℃, 300 rev/mins was cultivated described culture 16 hours.

30 milliliters of above-mentioned cultures are inoculated into 3 liters BSM-substratum (seeing embodiment 6e), then 7.5 liters fermentor tank (model Labfors, Infors AG, CH) in fermentation.Leavening temperature is 25 ℃, and the constant feed speed of gas is 3.2 liters/minute.Add glycerine solution [500 grams per liter glycerine, 10 milliliters/rise trace element solution, 10 milliliters/rise biotin solution (seeing embodiment 6e)] after 24 hours.During the fermentation, the speed of supply dosage progressively is increased to 45 milliliters/hour from 10 milliliters/hour.After 250 hours, under the streptokinase activation condition, can record the activity of the Profibrinolysin of 1375U/L.

Embodiment 8: the evaluation of plasminogen activator

Measured the activity of the plasminogen activation of 24 kinds of commercially available proteolytic enzyme.The system of experiment reaction is: the sodium phosphate buffer of 100mM pH=8.0,0.36mM CaCl ₂, 0.9%NaCl.

If the proteolytic enzyme that provides is powder, then be dissolved in the damping fluid; If solution then directly uses, can dilute with damping fluid as required.The Profibrinolysin solution (20mg/ml) of the protein enzyme solution of 25 μ l and 25 μ l of the present invention mixed be incorporated under 37 ℃ of conditions incubation 10 minutes.By the method for hydrolysis substrate N-tosyl-Gly-Pro-Lys-pNA, measure the activity of plasmin then.Concrete grammar is: the substrate solution [9.5 milligrams N-tosyl-Gly-Pro-Lys-pNA is dissolved in 75 milligrams of glycine/10 milliliter and 2% polysorbas20] of getting 200 μ l joins in the 850 μ l damping fluids, Profibrinolysin-protein enzyme solution with the previous incubation of 50 μ l mixes then, and continues incubation under 37 ℃ of conditions.The increase degree that consumes can be measured by the absorbance value of surveying under the 405nm.Consumption is because due to the proteolytic enzyme.When measuring consumption, use the damping fluid of similar preincubation and the mixture of proteolytic enzyme, replace the plasmin of preincubation and the mixture of proteolytic enzyme.

Streptomyces griseus (S.griseus) proteolytic enzyme, proteolytic enzyme VIII, proteolytic enzyme XXIII, proteolytic enzyme XIX, proteolytic enzyme XVIII, ficin, Zinc metalloproteinase, clostripain, Glu-C endoproteinase, proteolytic enzyme XIII, rotten papoid, Chymotrypsin, proteolytic enzyme X, bromeline, Padutin and protease A are all available from Sigma company, Deisenhofen; Trypsinase, papoid, Asp-N endoproteinase, neutral protease I, Lys-C endoproteinase, zymoplasm and elastoser be all available from Roche, the mannheim; Proteinase K is available from QIAGEN, Hilden.The concentration of each proteolytic enzyme storage liquid of preparation sees Table 3.Dilution factor F is indicated as and measures and with the multiple of proteolytic enzyme storage liquid dilution.

After the activation, can measure the activity (every milligram of protein of 1U/mg=per minute transforms the N-tosyl-Gly-Pro-Lys-pNA of 1 μ mol) of following plasmin:

Table 3:

Proteolytic enzyme	Plasmin activity after the activation	Protein concentration (mg/ml)	F
Proteolytic enzyme	Plasmin activity after the activation	Protein concentration (mg/ml)	F	The rotten papain chymotrypsin of streptomyces griseus protease protein enzyme VIII protease XXIII protease XIX protease XVIII ficin Zinc metalloproteinase clostripain Glu-C endoproteinase protease XIII protease X bromelain pancren protease A trypsase papain Asp-N endoproteinase neutral proteinase I Lys-C endoproteinase fibrin ferment elastoser Proteinase K	613.3U/mg 9U/mg * * 0.7U/mg 0.01U/mg 8.9U/mg 1.7U/mg 0.6U/mg 0.01U/mg * * * * * 0.02U/mg 11kU/mg * 4.3U/mg * * 83.0U/mg 0.63U/mg *	0.77 3.58 17.8 2.78 1.79 0.81 0.01 0.25 0.81 0.43 2.02 0.14 2.01 0.81 0.56 0.36 3.40 0.64 0.004 0.2 0.01 0.59 0.36 3.60	1000 1000 50000 100 100 1 1 1 1 1 1 1 1 1 1 1 100000 10 1 1 1 500 5 100

* for following enzyme, do not detect the activity of Profibrinolysin: proteolytic enzyme XXIII, proteolytic enzyme XIX, rotten papoid, Lys-C endoproteinase, Chymotrypsin, papoid, neutral protease I, proteolytic enzyme X, bromeline, Padutin and Proteinase K.

Embodiment 9: pharmaceutical preparation

Below the functional Profibrinolysin of used reorganization among these embodiment, all obtain by innovative approach of the present invention.According to this meaning, that " Profibrinolysin " refers to recombinate is little-, little-, Methionin-and L-glutamic acid Profibrinolysin, and " plasmin " finger protein hydrolysis cutting reorganization is little-, little-, Methionin-and L-glutamic acid Profibrinolysin and plasmin of obtaining.Can activate with identical plasminogen activator little-, little-, Methionin-and L-glutamic acid Profibrinolysin, plasminogen excited protein enzyme especially described above, but also be not limited to these examples; Activator and Profibrinolysin (little-, little-, Methionin-and the L-glutamic acid Profibrinolysin) ratio be 1: 1000 approximately.

Can pass through proteolysis method plasminogen activation, the i.e. proteolytic enzyme of mentioning in these present patent application: as the urokinase of tissue-type plasminogen activator, or proteolytic enzyme such as proteolytic enzyme VIII, streptomyces griseus proteolytic enzyme; Also can pass through and streptokinase or staphylokinase formation mixture, thus plasminogen activation.

Embodiment 9a: pharmaceutical preparation

Hydrogel

The basal component of hydrogel (100 gram)

Profibrinolysin 100U

Plasminogen activator 0.1U

Natvosol 10,000 3.5 grams

Selectable components (Sorbic Acid/potassium sorbate 0.1-0.4%, PHB ester 0.1%)

Pure water adds to 100.0 grams

Optionally, can use hypromellose (hypromellose) or methylcellulose gum to replace Natvosol respectively, the consumption of described hypromellose or methylcellulose gum is the 0.5-15.0 gram.

Gel

Profibrinolysin 1000U

Plasminogen activator 1U

Glycerine (85%) 150.0 gram

Natvosol 10,000 32.5 grams

Selectable components (Sorbic Acid/potassium sorbate 0.1-0.4%, PHB ester 0.1%)

Do not contain Lactated Ringer's solution and add to 1000.0 grams

Or:

100 gram gels comprise:

Profibrinolysin 100U

Plasminogen activator 0.1U

Natvosol 30,000 2.5 grams

Glycerine (85%) 10.0 gram

Selectable components (Sorbic Acid/potassium sorbate 0.1-0.2%, PHB ester 0.1%)

Pure water adds to 100.0 grams

Or:

100 gram gels comprise:

Profibrinolysin 100U

Plasminogen activator 0.1U

Poly vinylformic acid 1 gram

Propylene glycol 8 grams

Middle Polyglycerine three esters 8 grams

Diethylamine (regulating pH) is an amount of

Selectable components (Sorbic Acid/potassium sorbate 0.1-0.2%, PHB ester 0.1%)

2-propyl alcohol 0-1 gram

Water adds to 100 grams

Hydrophilic ointment (polyethylene glycol ointment)

50 grams comprise

Profibrinolysin 50U

Plasminogen activator 0.05U

Poly(oxyethylene glycol) 400 30.0 grams

Macrogol 4000 10.0 grams

Selectable components (Sorbic Acid/potassium sorbate 0.1-0.2%, PHB ester 0.1%)

Pure water adds to 50.0 grams

Or:

Anhydrous polyethylene glycol ointment

100 grams comprise

Profibrinolysin 100U

Plasminogen activator 0.1U

Liquid Macrogol 50 grams

Polyethylene glycol 1500 adds to 100 grams

Or:

Water absorbing ointment

Profibrinolysin 100U

Plasminogen activator 0.1U

Cetyl stearyl alcohol 29 grams

Paraffin, viscosity 34 grams

Vaseline, white 100 grams

Hydrophobic ointment

Profibrinolysin 100U

Plasminogen activator 0.1U

Vaseline 80.0 grams

Dilute liquid paraffin adds to 100 grams

Hydrophobic paste

Profibrinolysin 100U

Plasminogen activator 0.1U

Hypromellose 400 20 grams

Vaseline, white add to 100 grams

Or:

Profibrinolysin 100U

Plasminogen activator 0.1U

Carbomer (for example carbopol 974p) 15 grams

Paraffin, viscosity 40 grams

Vaseline, white add to 100 grams

Breast ester (creme)

Profibrinolysin 100U

Plasminogen activator 0.1U

Middle Polyglycerine three esters 20 grams

Emulsification cetyl stearyl alcohol 10 grams

Lanolin 10 grams

Sorbyl alcohol 10 grams

Selectable components (Sorbic Acid/potassium sorbate 0.1-0.2%, PHB ester 0.1%)

Pure water adds to 100 grams

Nonionic wetting ability butterfat

Profibrinolysin 100U

Plasminogen activator 0.1U

Soft ester alcohol 20 grams

2-myristic acid ethyl ester 10 grams

85% glycerine, 6 grams

Potassium sorbate 0.14 gram

Citric acid 0.07 gram

Water adds to 100 grams

Nonionic butterfat

Profibrinolysin 100U

Plasminogen activator 0.1U

Polysorbate 60 5 grams

Cetyl stearyl alcohol 10 grams

85% glycerine, 10 grams

Vaseline, white 25 grams

Selectable components (Sorbic Acid/potassium sorbate 0.1-0.2%, PHB ester 0.1%)

Water adds to 100 grams

Liposomal formulation

Profibrinolysin 100U

Plasminogen activator 0.1U

Soybean lecithin, ovum gallinaceum phosphatidase 11 5 grams

Selectable components (Sorbic Acid/potassium sorbate 0.1-0.2%, PHB ester 0.1% or diazonium biphenyl urea 1-2 gram)

Water adds to 100.0 grams

Capsule

The capsule of one 0.25 gram powder/granule comprises:

Profibrinolysin 5U

Plasminogen activator 0.005U

Starch 0.1 gram

Silicon-dioxide 0.02 gram

Magnesium Stearate 0.002 gram

Polymethacrylate copolyester/polymethyl acrylic acid 0.015 gram

Triethyl citrate 0.0005 gram

Talcum powder 0.001 gram

Micro-crystalline cellulose adds to 0.25 gram

Or:

The capsule of one 0.25 gram powder/granule comprises:

Profibrinolysin 5U

Plasminogen activator 0.005U

Silicon-dioxide 0.01 gram

Magnesium Stearate 0.002 gram

Polymethacrylate copolyester/polymethyl acrylic acid 0.015 gram

Triethyl citrate 0.0001 gram

0.001 milligram of talcum powder

N.F,USP MANNITOL adds to 0.25 gram

Pill

100 milligrams of granular pills comprise:

Profibrinolysin 5U

Plasminogen activator 0.005U

30 milligrams of starch

2 milligrams of silicon-dioxide

4 milligrams of Magnesium Stearates

5 milligrams of polymethacrylate copolyester/polymethyl acrylic acid

Triethyl citrate 0-1 milligram

0.00001 milligram of talcum powder

Micro-crystalline cellulose adds to 100 milligrams

Piller

100 gram pillers comprise

Profibrinolysin 2000U

Plasminogen activator 2U

Starch 20 grams

Stearic acid sucrose 20 grams

Silicon-dioxide 2 grams

Magnesium Stearate 3 grams

Polyvinylpyrrolidone 0-1 gram

Polymethacrylate copolyester/polymethyl acrylic acid 5 grams

Talcum powder 0.2 gram

Triethyl citrate 0.1 gram

Micro-crystalline cellulose adds to 100 grams

Injection

Profibrinolysin 500U

Plasminogen activator 0.5U

Ethanol 0-1 gram

Propylene glycol 10 grams

Polyoxyethylene glycol 0-1 gram

Sodium-chlor is an amount of

Selectable buffer liquid (Sodium phosphate dibasic/phosphate sodium dihydrogen buffer solution)

Pure water adds to 100 milliliters

In described preparation, can to use the plasmin that produces identical live vol to replace little-, little-, Methionin-or L-glutamic acid-Profibrinolysin.If directly use plasmin, then do not need to contain plasminogen activator so in the pharmaceutical preparation.

Embodiment 9b: pharmaceutical preparation

A) hydrogel

The basal component of hydrogel (100 gram)

Plasmin 100U

Natvosol 400 2.5-5 gram

Pure water adds to 100.0 grams

The expansion time needs 1 to 3 hour.

The plasmin that can contain 1-1000U in every gram hydrogel.

B) hydrophilic ointment

The essentially consist of hydrophilic ointment (1000 gram)

Plasmin 1000U

Anhydrous glycerol 85.0 grams

Natvosol 10,000 32.5 grams

Can select to add poly-Inositol Nicotinate (polyhexanide) 2 weight %

Do not contain Lactated Ringer's solution and add to 1000.0 grams

Poly-Inositol Nicotinate can be used as the active substance that suppresses microorganism growth and is optionally added, but is no more than 0.2% of gross weight.Can add Natvosol 400 (as: Tylose H 300 or Natrosol 250 HX PHARM) and replace Natvosol 10000 (Natrosol 250 HXPHARM).

Every gram ointment can contain the plasmin of 1-10000U.

C) ointment

The essentially consist of ointment (50 gram)

Plasmin 50U

Poly(oxyethylene glycol) 400 30.0-32.5 gram

Poly(oxyethylene glycol) 400 012.5-7.5 gram

Pure water adds to 50.0 grams

Preparation:

12.5 gram Macrogol 4000s and 30.0 gram poly(oxyethylene glycol) 400 (also can use 7.5 gram Macrogol 4000s and 32.5 gram poly(oxyethylene glycol) 400) are mixed and put into the ointment dish, and hot water adds bath and melts until polyoxyethylene glycol.After the cooling, can add the Profibrinolysin of producing according to the method for the invention in right amount, this Profibrinolysin has been dissolved in the 7.5 gram pure water, homogenizes then.

D) capsule

0.5 restrain capsular essentially consist

Profibrinolysin 5U

Lactose 0.42 gram

Starch 0.06 gram

Magnesium Stearate 0.02 gram

Each capsule can contain the plasmin of 0.1-100U.

E) injection/transfusion

The essentially consist of 100ml

Plasmin 500U

Ethanol 0.01 gram

30 milliliters of propylene glycol

Pure water adds to 100ml

Every ml soln can contain the plasmin of 1-500U.

Can with above-mentioned activity unit amount little-, little-, Methionin-, L-glutamic acid-Profibrinolysin replaces plasmin, if add at least a plasminogen activator simultaneously, the ratio of the plasminogen activator of described adding and Profibrinolysin should be between 1: 10000 to 1: 100, preferred 1: 1000, described amount was the live vol of Profibrinolysin.

Embodiment 10a: increase multi-form Miniplasminogen, Microplasminogen gene, and be cloned among the carrier pPICZaA; Transform pichia pastoris phaff.

Miniplasminogen, Microplasminogen are the derivatives shorter than total length Profibrinolysin, lack N end structure territory, but still have the activity that is activated into to plasmin.For with Miniplasminogen, Microplasminogen gene amplification and be cloned among the carrier pPICZ α A, 3 ' end primer NO34 and 5 ' different end primer NO36a-j (sequence number No.19 to 28) have been designed, the term harmonization described in the condition of amplification and the embodiment 1a.Except with the complementary base of Profibrinolysin gene, Oligonucleolide primers NO36a, c, e, j, i also have the codon of Kex2 restriction enzyme site; Primer NO36b, d, f, h, j have then added the codon of the restriction enzyme site of two Ste13 in the downstream of Kex2 restriction enzyme site codon.Primer NO34 goes up also has the KspI restriction enzyme site, and primer NO36 a-j then contains the XhoI restriction enzyme site.

With Miniplasminogen, the Microplasminogen gene clone method in the carrier pPICZ α A, similar with method among the embodiment 1b, just carrier and specific PCR product all use restriction enzyme XhoI and KspI to come enzyme to cut.Title, encoded protein restriction enzyme site, the title of gained plasmid and the title of oozy Profibrinolysin derivative N terminal amino acid of the primer, the Profibrinolysin derivative that obtains all are summarized in following table.

5 '-primer	3 '-primer	Title	Proteolytic enzyme-restriction enzyme site	The plasmid title	N terminal amino acid *
5 '-primer	3 '-primer	Title	Proteolytic enzyme-restriction enzyme site	The plasmid title	N terminal amino acid *	NO36a NO36b NO36c NO36d NO36e NO36f NO36g NO36h NO36i NO36j	NO34 NO34 NO34 NO34 NO34 NO34 NO34 NO34 NO34 NO34	Miniplasminogen Miniplasminogen Microplasminogen Microplasminogen Microplasminogen Microplasminogen Microplasminogen Microplasminogen Microplasminogen Microplasminogen	Kex2 Kex2; 2 Ste13 Kex2 Kex2; 2 Ste13 Kex2 Kex2; 2 Ste13 Kex2 Kex2; 2 Ste13 Kex2 Kex2,2 Ste13	pPLG1.1 pPLG2.1 pPLG3.2 pPLG4.2 pPLG5.3 pPLG6.1 pPLG7.1 pPLG8.3 pPLG9.1 pPLG10.1	A463 A463 K550 K550 L551 L551 A562 A562 S564 S564

* refer to the preceding Profibrinolysin (Seq.ID No.12) that constitutes by 810 amino acid.

Fig. 8 has shown example: plasmid pPLG1.1.

As transforming the pichia pastoris phaff KM71H strain method among the embodiment 1c with pMHS476.1, transform pichia pastoris phaff KM71H strain with pPLG1.1, the clone's called after pichia pastoris phaff KM71H/pPLG1.1-1/a that obtains, " a " still represents the serial number since 1 clone herein.

According to the method that produces the KM71H/pPLG1.1-1/a bacterial strain, utilize following plasmid to produce different bacterial strains: pPLG2.1, pPLG3.2, pPLG4.2, pPLG5.3, pPLG6.1, pPLG7.1, pPLG8.3, pPLG9.1 or pPLG10.1.

Oligonucleolide primers NO36a-j:

NO36a AAAAACTCGAGAAAAGAGCACCTCCGCCTGTTG

NO36b AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTG

NO36c AAAAACTCGAGAAAAGAAAACTTTACGACTACTG

NO36d AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTG

NO36e AAAAACTCGAGAAAAGACTTTACGACTACTGTG

NO36f AAAAACTCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTG

NO36g AAAAACTCGAGAAAAGAGCCCCTTCATTTGATTGTG

NO36h AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTG

NO36i AAAAACTCGAGAAAAGATCATTTGATTGTGGGAAGCC

NO36j AAAAACTCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCC

Embodiment 10b: increase multi-form little-, little-the Profibrinolysin gene, and be cloned among the carrier pGAPZaA; It is transformed pichia pastoris phaff.

For with little-, little-Profibrinolysin gene amplification and being cloned among the carrier pGAPZ α A, designed 3 ' end primer NO34 and 5 ' different end primer NO36a-j (Seq.ID No.19 to 28), the term harmonization described in the condition of amplification and the embodiment 1a.Oligonucleolide primers NO36a, c, e, j, i are at the other codon with Kex2 restriction enzyme site of the complementary base of Profibrinolysin gene; Primer NO36b, d, f, h, j have then added the codon of the restriction enzyme site of two Ste13 in the downstream of Kex2 restriction enzyme site codon.Primer NO34 goes up also has the KspI restriction enzyme site, and primer NO36a-j then contains the XhoI restriction enzyme site.

With little-, little-Profibrinolysin gene clone is to the method for carrier pGAPZ α A, similar with method among the embodiment 1b, just carrier and PCR product all use restriction enzyme XhoI and KspI enzyme to cut.The title of the title of the title of the primer, Profibrinolysin derivative, encoded protein restriction enzyme site, gained plasmid and oozy Profibrinolysin derivative N terminal amino acid all is summarized in following table.

5 '-primer	3 '-primer	Title	Proteolytic enzyme-restriction enzyme site	The plasmid title	N terminal amino acid *
5 '-primer	3 '-primer	Title	Proteolytic enzyme-restriction enzyme site	The plasmid title	N terminal amino acid *	NO36a NO36b NO36c NO36d NO36e NO36f NO36g NO36h NO36i NO36j	NO34 NO34 NO34 NO34 NO34 NO34 NO34 NO34 NO34 NO34	Miniplasminogen Miniplasminogen Microplasminogen Microplasminogen Microplasminogen Microplasminogen Microplasminogen Microplasminogen Microplasminogen Microplasminogen	Kex2 Kex2,2 Ste13 Kex2 Kex2,2 Ste13 Kex2 Kex2,2 Ste13 Kex2 Kex2,2 Ste13 Kex2 Kex2,2 Ste13	pPLG11.2 pPLG11.2 pPLG13.1 pPLG14.2 pPLG15.1 pPLG16.3 pPLG17.2 pPLG18.1 pPLG19.2 pPLG20.1	A463 A463 K550 K550 L551 L551 A562 A562 S564 S564

Fig. 9 has shown example: plasmid pPLG11.2.

As transforming the method for pichia pastoris phaff KM71H strain with pJW9.1 among the embodiment 7a, transform pichia pastoris phaff KM71H strain with pPLG11.2, cut plasmid with restriction endonuclease BlnI earlier before transforming and makes it linearizing.Clone's called after pichia pastoris phaff KM71H/pPLG11.2-1/a of gained, " a " still represents the serial number since 1 clone herein.

According to the method that produces the KM71H/pPLG1.1-1/a bacterial strain, utilize following plasmid to produce different bacterial strains: pPLG12.1, pPLG13.1, pPLG14.2, pPLG15.1, pPLG16.3, pPLG17.2, pPLG18.1, pPLG19.2 and pPLG20.1.

Sequential recording

Sequence 1: Oligonucleolide primers NO34

AAAAACCGCGGTCAATTATTTCTCATCACTCCC

Sequence 2: Oligonucleolide primers NO36

AAAAACTCGAGAAAAGAAAAGTGTATCTCTCAGAGTG

Sequence 3: Oligonucleolide primers NO57

AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAAGTGTATCTCTCAGAGTG

Sequence 4: Oligonucleolide primers NO37

AAAAATTCGAAAAATGGAACATAAGGAAGTGG

Sequence 5: Oligonucleolide primers NO35

AAAAACTCGAGAAAAGAGAGCCTCTGGATGACTAT

Sequence 6: Oligonucleolide primers NO56

AAAAACTCGAGAAAAGAGAGGCTGAAGCTGAGCCTCTGGATGACTAT

Sequence 7: people's Methionin Profibrinolysin fusion gene, it contains the password of Kex2 protease cutting site

The gene of the signal sequence of son and yeast saccharomyces cerevisiae alpha factor

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT

CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT

TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT

AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA

TCTCTCGAGAAAAGAAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTAC

AGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCT

CCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTAC

TGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAG

AGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAA

AACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCT

CAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAG

AATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAAC

AAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCC

ACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTT

TCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCA

GAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGG

GCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCC

TGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACC

CCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACC

ACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAG

ACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCC

GATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTG

AAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCA

GATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGC

AAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCAT

AGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGC

CGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTT

TACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAA

GTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCC

TGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTG

ATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCA

TCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAA

ATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTA

AGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTAT

GTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTT

GGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGC

TATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGA

GGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAA

TACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTz

GTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 8: people's Methionin Profibrinolysin fusion rotein, it contains the signal peptide of Kex2 protease cutting site and yeast saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRKVYLSECKTGNGKNY

RGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQG

PWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDS

QSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPR

CTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTP

ENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQ

LAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQK

TPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASV

VAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPH

RHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCA

APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTL

ISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEP

TRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTF

GAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSG

GPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 9: people's Methionin Profibrinolysin fusion gene, it contains the codon of Kex2 protease cutting site, two Ste3 protease cutting site and the signal peptide sequence of yeast saccharomyces cerevisiae alpha factor

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT

CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT

TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT

AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA

TCTCTCGAGAAAAGAGAGGCTGAAGCTAAAGTGTATCTCTCAGAGTGCAAGACTGGGAAT

GGAAAGAACTACAGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGG

AGTTCCACTTCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTG

GAGGAGAACTACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACT

GATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCAT

TGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAG

GCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAG

AACCTGAAGAAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACC

ACCGACCCCAACAAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCA

TCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTG

GCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACAT

AACAGGACACCAGAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCT

GACGGAAAAAGGGCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGT

AAGATACCGTCCTGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCA

CCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGC

ACATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACAC

CGGCACCAGAAGACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGG

AATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAG

TACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTT

GTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAA

GGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCC

CAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAA

AAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAAT

CCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGT

GGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCC

CACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGT

GGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCC

CCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCG

CATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCC

TTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCA

TCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACC

CAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAA

GTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGG

CATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTC

GAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCC

AATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATG

AGAAATAATTGA

Sequence 10: people's Methionin Profibrinolysin fusion rotein, it contains the signal peptide of Ste3, Kex2 protease cutting site and yeast saccharomyces cerevisiae alpha factor.

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAKVYLSECKTGN

GKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDN

DPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQ

AWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELC

DIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTH

NRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPV

STEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPH

RHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGT

EASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAA

QEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDV

PQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFC

GGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRL

FLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGET

QGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQ

GDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVM

RNN*

Sequence 11: Profibrinolysin gene before the people

ATGGAACATAAGGAAGTGGTTCTTCTACTTCTTTTATTTCTGAAATCAGGTCAAGGAGAG

CCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTCAGTGTCACTAAGAAGCAG

CTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTCACC

TGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAACAGG

AAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAAAAGAAAGTGTATCTC

TCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAACAAAAAAT

GGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGATTCTCACCTGCT

ACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCGCAG

GGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAG

TGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACC

ATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATT

CCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGGGAG

CTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTTTGCGACATCCCC

CGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACAGGT

GAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGGAGT

GCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGCAAAAATTTGGAT

GAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCATACAACCAACAGC

CAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCAGTATCCACGGAA

CAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGT

GATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCT

TGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCTGGC

CTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACCACA

GACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGAGT

GTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGAC

TGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACG

CCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACA

AATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGT

CCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGT

GCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGG

GTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACA

AGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCT

GCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACAC

CAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAG

CCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAA

GTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTC

ATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAG

CTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAA

TCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGT

GGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGG

GGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTT

ACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 12: Profibrinolysin before the people

MEHKEVVLLLLLFLKSGQGEPLDDYVNTQGASLFSVTKKQLGAGSIEECA

AKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSIIIRMRDVVLFEKKVYL

SECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEGLEEN

YCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGKISKT

MSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDP

NKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTCQHWS

AQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEYCKIP

SCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGKKCQS

WSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEYCN

LKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGT

PCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRK

LYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRT

RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQ

EIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECF

ITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLA

GGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFV

TWIEGVMRNN*

Sequence 13: human glutamic acid Profibrinolysin fusion gene, it contains the codon of Kex2 protease cutting site and the signal sequence of yeast saccharomyces cerevisiae alpha factor

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT

CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT

TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT

AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA

TCTCTCGAGAAAAGAGAGCCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTC

AGTGTCACTAAGAAGCAGCTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAG

GAGGACGAAGAATTCACCTGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTG

ATAATGGCTGAAAACAGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTT

GAAAAGAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACG

ATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGA

CCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAAT

CCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGAC

TACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGAC

GGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCA

CACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGT

CGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGG

GAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAG

TGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCAC

ACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTC

CCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGG

TGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCC

TCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTC

CAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACA

GGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAA

AACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGC

CCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGC

TCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAG

ACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCG

ACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGC

ATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCT

GATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTAC

TGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCG

AAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGG

CAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCA

GAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAG

GTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTG

TCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCT

GCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCT

GACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGC

CTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTT

CTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGAC

AGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTA

CAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTT

CGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 14: human glutamic acid Profibrinolysin fusion rotein, it contains the signal peptide of Kex2 protease cutting site and yeast saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREPLDDYVNTQGASLF

SVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENRKSSI

IIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHR

PRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEE

CMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYC

RNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYR

GNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPW

CHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQS

YRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKG

PWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFG

NGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNP

DGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGG

CVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYK

VILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPA

CLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEF

LNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGC

ARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 15: human glutamic acid Profibrinolysin fusion gene, it contains the codon of Kex2, two Ste13 protease cutting site and the signal peptide gene of yeast saccharomyces cerevisiae alpha factor

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCT

CCAGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGT

TACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAAT

AACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTA

TCTCTCGAGAAAAGAGAGGCTGAAGCTGAGCCTCTGGATGACTATGTGAATACCCAGGGG

GCTTCACTGTTCAGTGTCACTAAGAAGCAGCTGGGAGCAGGAAGTATAGAAGAATGTGCA

GCAAAATGTGAGGAGGACGAAGAATTCACCTGCAGGGCATTCCAATATCACAGTAAAGAG

CAACAATGTGTGATAATGGCTGAAAACAGGAAGTCCTCCATAATCATTAGGATGAGAGAT

GTAGTTTTATTTGAAAAGAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAAC

TACAGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACT

TCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAAC

TACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAA

AAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGA

GAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGAC

TCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAG

AAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCC

AACAAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGT

CCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACC

GTTTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACA

CCAGAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAA

AGGGCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCG

TCCTGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTA

ACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCC

ACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAG

AAGACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGAT

GCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAAC

CTGAAAAAATGCTCAGGAACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTT

CCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGA

GGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCC

CATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTAC

TGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAA

CTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCT

CAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACAT

TCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACC

TTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCT

TCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAG

GAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAG

CTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAAT

TATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACT

TTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAAT

CGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCC

GGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGAC

AAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCT

GGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAAT

TGA

Sequence 16: human glutamic acid Profibrinolysin fusion rotein, it contains the signal peptide of Kex2, Ste13 restriction enzyme site and yeast saccharomyces cerevisiae alpha factor

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAEPLDDYVNTQG

ASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSKEQQCVIMAENR

KSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTKNGITCQKWSST

SPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILE

CEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLK

KNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTG

ENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGK

RAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHG

DGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPD

ADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVLLPDVETPSEED

CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNY

CRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGR

VVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRP

SSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDK

VIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCN

RYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSW

GLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 17: the sequence (pSM49.8, pSM58.1 and pSM82.1) that is secreted into the L-glutamic acid Profibrinolysin in the substratum

EPLDDYVNTQGASLFSVTKKQLGAGSIEECAAKCEEDEEFTCRAFQYHSK

EQQCVIMAENRKSSIIIRMRDVVLFEKKVYLSECKTGNGKNYRGTMSKTK

NGITCQKWSSTSPHRPRFSPATHPSEGLEENYCRNPDNDPQGPWCYTTDP

EKRYDYCDILECEEECMHCSGENYDGKISKTMSGLECQAWDSQSPHAHGY

IPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWELCDIPRCTTPPPSS

GPTYQCLKGTGENYRGNVAVTVSGHTCQHWSAQTPHTHNRTPENFPCKNL

DENYCRNPDGKRAPWCHTTNSQVRWEYCKIPSCDSSPVSTEQLAPTAPPE

LTPVVQDCYHGDGQSYRGTSSTTTTGKKCQSWSSMTPHRHQKTPENYPNA

GLTMNYCRNPDADKGPWCFTTDPSVRWEYCNLKKCSGTEASVVAPPPVVL

LPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPE

TNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGK

PQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLT

AAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALL

KLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEA

QLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEK

DKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 18: the protein sequence (pMHS476.1, pSM54.2, pAC37.1 and pJW9.1) that is secreted into the Methionin Profibrinolysin in the substratum.

KVYLSECKTGNGKNYRGTMSKTKNGITCQKWSSTSPHRPRFSPATHPSEG

LEENYCRNPDNDPQGPWCYTTDPEKRYDYCDILECEEECMHCSGENYDGK

ISKTMSGLECQAWDSQSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCF

TTDPNKRWELCDIPRCTTPPPSSGPTYQCLKGTGENYRGNVAVTVSGHTC

QHWSAQTPHTHNRTPENFPCKNLDENYCRNPDGKRAPWCHTTNSQVRWEY

CKIPSCDSSPVSTEQLAPTAPPELTPVVQDCYHGDGQSYRGTSSTTTTGK

KCQSWSSMTPHRHQKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRW

EYCNLKKCSGTEASVVAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATT

VTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTT

NPRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQV

SLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLE

PHVQEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADR

TECFITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCA

GHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRV

SRFVTWIEGVMRNN*

Sequence 19: Oligonucleolide primers NO36a

AAAAACTCGAGAAAAGAGCACCTCCGCCTGTTG

Sequence 20: Oligonucleolide primers NO36b

AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCACCTCCGCCTGTTG

Sequence 21: Oligonucleolide primers NO36c

AAAAACTCGAGAAAAGAAAACTTTACGACTACTG

Sequence 22: Oligonucleolide primers NO36d

AAAAACTCGAGAAAAGAGAGGCTGAAGCTAAACTTTACGACTACTG

Sequence 23: Oligonucleolide primers NO36e

AAAAACTCGAGAAAAGACTTTACGACTACTGTG

Sequence 24: Oligonucleolide primers NO36f

AAAAACTCGAGAAAAGAGAGGCTGAAGCTCTTTACGACTACTGTG

Sequence 25: Oligonucleolide primers NO36g

AAAAACTCGAGAAAAGAGCCCCTTCATTTGATTGTG

Sequence 26: Oligonucleolide primers NO36h

AAAAACTCGAGAAAAGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTG

Sequence 27: Oligonucleolide primers NO36i

AAAAACTCGAGAAAAGATCATTTGATTGTGGGAAGCC

Sequence 28: Oligonucleolide primers NO36j

AAAAACTCGAGAAAAGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCC

Sequence 29: Miniplasminogen (pPLG1.1 and pPLG2.1)

APPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHR

HSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAA

PSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLI

SPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPT

RKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFG

AGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGG

PLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 30: Microplasminogen (pPLG3.2 and pPLG4.2)

KLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLR

TRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHV

QEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTEC

FITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHL

AGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRF

VTWIEGVMRNN*

Sequence 31: Microplasminogen (pPLG5.3 and pPLG6.1).

LYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRT

RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQ

EIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECF

ITGWGETQGTFGAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLA

GGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFV

TWIEGVMRNN*

Sequence 32: Microplasminogen (pPLG7.1 and pPLG8.3).

APSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTL

ISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEP

TRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTF

GAGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSG

GPLVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 33: Microplasminogen (pPLG9.1 and pPLG10.1).

SFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLIS

PEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTR

KDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGA

GLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGP

LVCFEKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

The dna sequence dna of the yeast saccharomyces cerevisiae alpha factor among the sequence 34:pPICZ α A, this sequence is positioned at the upstream of Kex2 restriction enzyme site

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAG

The aminoacid sequence of the yeast saccharomyces cerevisiae alpha factor among the sequence 35:pPICZ α A, described sequence is positioned at the upstream of Kex2 restriction enzyme site

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLE

The dna sequence dna of sequence 36:Kex2 restriction enzyme site

AAAAGA

The dna sequence dna of sequence 37:Ste13 restriction enzyme site

GAGGCTGAAGCT

The aminoacid sequence of sequence 38:Kex2 restriction enzyme site

KR

The aminoacid sequence of sequence 39:Ste13 restriction enzyme site

EAEA

Sequence 40: the aminoacid sequence of being made up of following sequence: the propetide of the people's Miniplasminogen among the plasmid pPLG1.1, Kex2 restriction enzyme site and yeast saccharomyces cerevisiae alpha factor is former

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKRAPPPVVLLPDVETPS

EEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLE

KNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVEPKKC

PGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKS

PRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSSPAVI

TDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENK

VCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGV

TSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 41: the aminoacid sequence of being made up of following sequence: the propetide of the people's Miniplasminogen among the plasmid pPLG2.1, Kex2 restriction enzyme site, two Ste1 restriction enzyme sites and yeast saccharomyces cerevisiae alpha factor is former

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDV

AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEAAPPPVVLLPDV

ETPSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPR

AGLEKNYCRNPDGDVGGPWCYTTNPRKLYDYCDVPQCAAPSFDCGKPQVE

PKKCPGRVVGGCVAHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHC

LEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIALLKLSS

PAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPV

IENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYI

LQGVTSWGLGCARPNKPGVYVRVSRFVTWI EGVMRNN*

Sequence 42: the aminoacid sequence of being made up of following sequence: the propetide of the people's Microplasminogen among the plasmid pPLG3.2, Kex2 restriction enzyme site and yeast saccharomyces cerevisiae alpha factor is former

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKRKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPH

SWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIE

VSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLL

KEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTS

WGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 43: the aminoacid sequence of being made up of following sequence: the propetide of the people's Microplasminogen among the plasmid pPLG4.2, Kex2 restriction enzyme site, two Ste13 restriction enzyme sites and yeast saccharomyces cerevisiae alpha factor is former

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKREAEAKLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCV

AHPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHV

QEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFG

AGLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQ

GVTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 44: the aminoacid sequence of being made up of following sequence: the propetide of the people's Microplasminogen among the plasmid pPLG5.3, Kex2 restriction enzyme site and yeast saccharomyces cerevisiae alpha factor is former

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKRLYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHS

WPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEV

SRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLK

EAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSW

GLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 45: the aminoacid sequence of being made up of following sequence: the propetide of the people's Microplasminogen among the plasmid pPLG6.1, Kex2 restriction enzyme site, two Ste13 restriction enzyme sites and yeast saccharomyces cerevisiae alpha factor is former

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKREAEALYDYCDVPQCAAPSFDCGKPQVEPKKCPGRVVGGCVA

HPHSWPWQVSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQ

EIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGA

GLLKEAQLPVIENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQG

VTSWGLGCARPNKPGVYVRVSRFVTWIEGVMRNN*

Sequence 46: the aminoacid sequence of being made up of following sequence: the propetide of the people's Microplasminogen among the plasmid pPLG7.1, Kex2 restriction enzyme site and yeast saccharomyces cerevisiae alpha factor is former

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKRAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRF

GMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKD

IALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKV

CNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPG

VYVRVSRFVTWIEGVMRNN*

Sequence 47: the aminoacid sequence of being made up of following sequence: the propetide of the people's Microplasminogen among the plasmid pPLG8.3, Kex2 restriction enzyme site, two Ste13 restriction enzyme sites and yeast saccharomyces cerevisiae alpha factor is former

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKREAEAAPSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSL

RTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEP

TRKDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVI

ENKVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARP

NKPGVYVRVSRFVTWIEGVMRNN*

Sequence 48: the aminoacid sequence of being made up of following sequence: the propetide of the people's Microplasminogen among the plasmid pPLG9.1, Kex2 restriction enzyme site and yeast saccharomyces cerevisiae alpha factor is former

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKRSFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRTRFGM

HFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTRKDIA

LLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIENKVCN

RYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNKPGVY

VRVSRFVTWIEGVMRNN*

Sequence 49: the aminoacid sequence of being made up of following sequence: the propetide of the people's Microplasminogen among the plasmid pPLG10.1, Kex2 restriction enzyme site, two Ste13 restriction enzyme sites and yeast saccharomyces cerevisiae alpha factor is former

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGL

LFINTTIASIAAKEEGVSLEKREAEASFDCGKPQVEPKKCPGRVVGGCVAHPHSWPWQVSLRT

RFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAHQEVNLEPHVQEIEVSRLFLEPTR

KDIALLKLSSPAVITDKVIPACLPSPNYVVADRTECFITGWGETQGTFGAGLLKEAQLPVIEN

KVCNRYEFLNGRVQSTELCAGHLAGGTDSCQGDSGGPLVCFEKDKYILQGVTSWGLGCARPNK

PGVYVRVSRFVTWIEGVMRNN*

Sequence 50: the nucleotide sequence of forming by following sequence: the people's Miniplasminogen gene among the plasmid pPLG1.1, the codon of Kex2 restriction enzyme site and the former gene of yeast saccharomyces cerevisiae alpha factor propetide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATG

TTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAG

GACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCG

GGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACG

ACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGAT

TGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCC

CACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGA

GGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGG

CCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAG

GAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTA

AGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTG

GTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCT

GGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTT

CTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGT

TGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGA

GTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCA

AGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 51: the nucleotide sequence of forming by following sequence: the people's Miniplasminogen gene among the plasmid pPLG2.1, the codon of Kex2 restriction enzyme site and Ste13 restriction enzyme site, and the former gene of yeast saccharomyces cerevisiae alpha factor propetide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGAGGCTGAAGCTGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAA

GAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGG

ACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACA

AATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCC

TGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCC

CCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGG

GGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATG

CACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAG

AAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAA

CCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCC

TTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCC

CCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGT

ACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAAT

CGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGA

GGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATAC

ATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTAT

GTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 52: the nucleotide sequence of forming by following sequence: the people's Microplasminogen gene among the plasmid pPLG3.2, the codon of Kex2 restriction enzyme site and the former gene of yeast saccharomyces cerevisiae alpha factor propetide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAG

CCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACAT

TCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTG

ATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCC

TACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAA

GTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCT

GCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGAC

CGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTC

AAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGA

AGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGT

GACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCT

TGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTT

ACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 53: the nucleotide sequence of forming by following sequence: the people's Microplasminogen gene among the plasmid pPLG4.2, the codon of Kex2 restriction enzyme site and Ste13 restriction enzyme site, and the former gene of yeast saccharomyces cerevisiae alpha factor propetide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGAGGCTGAAGCTAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTT

GATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTG

GCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGT

GGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCA

AGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTT

CAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAG

CTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTAT

GTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGA

GCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAG

TTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGAC

AGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAA

GGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTT

TCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 54: the nucleotide sequence of forming by following sequence: the people's Microplasminogen gene among the plasmid pPLG5.3, the codon of Kex2 restriction enzyme site and the former gene of yeast saccharomyces cerevisiae alpha factor propetide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCT

CAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCC

TGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATA

TCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTAC

AAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTG

TCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCC

GTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGG

ACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAG

GAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGA

GTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGAC

AGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGG

GGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACT

TGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 55: the nucleotide sequence of forming by following sequence: the people's Microplasminogen gene among the plasmid pPLG6.1, the codon of Kex2 restriction enzyme site and Ste13 restriction enzyme site, and the former gene of yeast saccharomyces cerevisiae alpha factor propetide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGAGGCTGAAGCTCTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGAT

TGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCC

CACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGA

GGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGG

CCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAG

GAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTA

AGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTG

GTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCT

GGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTT

CTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGT

TGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGA

GTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCA

AGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 56: the nucleotide sequence of forming by following sequence: the people's Microplasminogen gene among the plasmid pPLG7.1, the codon of Kex2 restriction enzyme site and the former gene of yeast saccharomyces cerevisiae alpha factor propetide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTT

GTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTT

GGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGC

TTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAAT

CTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGAT

ATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTG

CCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACC

CAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTG

TGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTG

GCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGAC

AAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGT

GTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 57: the nucleotide sequence of forming by following sequence: the codon of people's Microplasminogen gene, Kex2 restriction enzyme site and Ste13 restriction enzyme site among the plasmid pPLG8.3, and the former gene of yeast saccharomyces cerevisiae alpha factor propetide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGAGGCTGAAGCTGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGT

CCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTT

AGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACT

GCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACAC

CAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCC

ACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATC

CCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGC

TGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATT

GAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGT

GCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGC

TTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCC

AATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGA

AATAATTGA

Sequence 58: the nucleotide sequence of forming by following sequence: the people's Microplasminogen gene among the plasmid pPLG9.1, the codon of Kex2 restriction enzyme site and the former gene of yeast saccharomyces cerevisiae alpha factor propetide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGATCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGG

GGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATG

CACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAG

AAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAA

CCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCC

TTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCC

CCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGT

ACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAAT

CGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGA

GGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATAC

ATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTAT

GTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 59: the nucleotide sequence of forming by following sequence: the codon of people's Microplasminogen gene, Kex2 restriction enzyme site and Ste13 restriction enzyme site among the plasmid pPLG10.1, and the former gene of yeast saccharomyces cerevisiae alpha factor propetide

ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCA

GTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCA

GATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTA

TTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGAGAAA

AGAGAGGCTGAAGCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGA

AGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACA

AGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCC

CACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAA

GTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGA

AAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCT

TGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGA

GAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAAT

AAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGG

CATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAG

AAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAG

CCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAAT

TGA

Sequence 60: the nucleotide sequence of the people's Miniplasminogen gene among plasmid pPLG1.1 and the pPLG2.1.

GCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTT

GGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGAC

TGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAATCCACGGGCGGGT

CTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACA

AATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGT

GGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCAC

CCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGC

ACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCT

TCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAA

ATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGC

AGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTC

GCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGC

CTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTG

AATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGC

CAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTC

ACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGG

TTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 61: the nucleotide sequence of the people's Microplasminogen gene among plasmid pPLG3.2 and the pPLG4.2

AAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCT

CAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCC

TGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATA

TCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTAC

AAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTG

TCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCC

GTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGG

ACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAG

GAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGA

GTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGAC

AGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGG

GGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACT

TGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 62: the nucleotide sequence of the people's Microplasminogen gene among plasmid pPLG5.3 and the pPLG6.1

CTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAA

GTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGG

CCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCC

CCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAG

GTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCT

AGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTC

ATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACC

GAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAA

GCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTC

CAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGACAGT

GGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCTTGGGGT

CTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTTGTTACTTGG

ATTGAGGGAGTGATGAGAAATAATTGA

Sequence 63: the nucleotide sequence of the people's Microplasminogen gene among plasmid pPLG7.1 and the pPLG8.3

GCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTG

GGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGA

ATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTG

GAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTC

GAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATT

GCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCA

TCCCCAAATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAA

GGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGC

AATCGCTATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCC

GGAGGCACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAA

TACATTTTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTC

TATGTTCGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 64: the nucleotide sequence of the people's Microplasminogen gene among plasmid pPLG9.1 and the pPLG10.1

TCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGAAGGGTTGTGGGGGGG

TGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGAACAAGGTTTGGAATGCAC

TTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACTGCTGCCCACTGCTTGGAGAAG

TCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCACACCAAGAAGTGAATCTCGAACCG

CATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTG

CTAAAGCTAAGCAGTCCTGCCGTCATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCA

AATTATGTGGTCGCTGACCGGACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACT

TTTGGAGCTGGCCTTCTCAAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGC

TATGAGTTTCTGAATGGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGC

ACTGACAGTTGCCAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATT

TTACAAGGAGTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTT

CGTGTTTCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 65: the nucleotide sequence of human glutamic acid Profibrinolysin gene

GAGCCTCTGGATGACTATGTGAATACCCAGGGGGCTTCACTGTTCAGTGTCACTAAGAAG

CAGCTGGGAGCAGGAAGTATAGAAGAATGTGCAGCAAAATGTGAGGAGGACGAAGAATTC

ACCTGCAGGGCATTCCAATATCACAGTAAAGAGCAACAATGTGTGATAATGGCTGAAAAC

AGGAAGTCCTCCATAATCATTAGGATGAGAGATGTAGTTTTATTTGAAAAGAAAGTGTAT

CTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAACAAAA

AATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGATTCTCACCT

GCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCG

CAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTT

GAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAG

ACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGGATAC

ATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGG

GAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTTTGCGACATC

CCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACA

GGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGG

AGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGCAAAAATTTG

GATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCATACAACCAAC

AGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCAGTATCCACG

GAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCAT

GGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAG

TCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCT

GGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACC

ACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCG

AGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCTTCCGAAGAA

GACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACTGTTACTGGG

ACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAG

ACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAGGT

GGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGATGTCCCTCAG

TGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAATGTCCTGGA

AGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTCAGTCTTAGA

ACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGGGTGTTGACT

GCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATCCTGGGTGCA

CACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGGCTGTTCTTG

GAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTCATCACTGAC

AAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGGACCGAATGT

TTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTCAAGGAAGCC

CAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAATGGAAGAGTC

CAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGCCAGGGTGAC

AGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGAGTCACTTCT

TGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTTTCAAGGTTT

GTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Sequence 66: the nucleotide sequence of people's Methionin Profibrinolysin gene

AAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCC

AAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGA

TTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGAC

AACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGC

GACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAA

ATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCT

CATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAAC

CCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTT

TGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTG

AAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGT

CAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGC

AAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCAT

ACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCA

GTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGAC

TGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAG

AAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTAC

CCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGG

TGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGA

ACAGAAGCGAGTGTTGTAGCACCTCCGCCTGTTGTCCTGCTTCCAGATGTAGAGACTCCT

TCCGAAGAAGACTGTATGTTTGGGAATGGGAAAGGATACCGAGGCAAGAGGGCGACCACT

GTTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTC

ACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGT

GATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGAT

GTCCCTCAGTGTGCGGCCCCTTCATTTGATTGTGGGAAGCCTCAAGTGGAGCCGAAGAAA

TGTCCTGGAAGGGTTGTGGGGGGGTGTGTGGCCCACCCACATTCCTGGCCCTGGCAAGTC

AGTCTTAGAACAAGGTTTGGAATGCACTTCTGTGGAGGCACCTTGATATCCCCAGAGTGG

GTGTTGACTGCTGCCCACTGCTTGGAGAAGTCCCCAAGGCCTTCATCCTACAAGGTCATC

CTGGGTGCACACCAAGAAGTGAATCTCGAACCGCATGTTCAGGAAATAGAAGTGTCTAGG

CTGTTCTTGGAGCCCACACGAAAAGATATTGCCTTGCTAAAGCTAAGCAGTCCTGCCGTC

ATCACTGACAAAGTAATCCCAGCTTGTCTGCCATCCCCAAATTATGTGGTCGCTGACCGG

ACCGAATGTTTCATCACTGGCTGGGGAGAAACCCAAGGTACTTTTGGAGCTGGCCTTCTC

AAGGAAGCCCAGCTCCCTGTGATTGAGAATAAAGTGTGCAATCGCTATGAGTTTCTGAAT

GGAAGAGTCCAATCCACCGAACTCTGTGCTGGGCATTTGGCCGGAGGCACTGACAGTTGC

CAGGGTGACAGTGGAGGTCCTCTGGTTTGCTTCGAGAAGGACAAATACATTTTACAAGGA

GTCACTTCTTGGGGTCTTGGCTGTGCACGCCCCAATAAGCCTGGTGTCTATGTTCGTGTT

TCAAGGTTTGTTACTTGGATTGAGGGAGTGATGAGAAATAATTGA

Reference

(1)=and Desire Collen, thrombosis and hemostasis (Thrombosis and

Haemostasis)》，82，1999

(2)=Forsgren etc.. European biological chemistry association's federation's wall bulletin (FEBS Lett.)

213，1987

(3)=Petersen etc., biology and The Chemicals (J.Biol.Chem.), 265,

1990

(4)=Duman etc., biotechnology and applied biochemistry (Biotechnol.Appl.

Biochem.)28；39-45，1998

(5)=Guan etc., biotechnology journal (Sheng Wu Gong Cheng Xue Bao),

17，2001

(6)=Gonzalez-Gronow etc., Biochimica et Biophysica Acta,

1039，1990

(7)=Whitefleet-Smith etc., biological chemistry and biophysics document (Arch.

Biochem.Biophys.)，271，1989

(8)=and Nilsen und Castellino, protein expression and purifying (Protein

Expression and Purification)，16，1999

(9)=Busby etc., biology and The Chemicals (J.Biol.Chem.), 266,1991

(10)=Maniatis etc., molecular cloning: laboratory manual (Molecular Cloning:

A Laboratory Manual), (the Cold Spring Harbor of cold spring port press

press)，1989

(11)＝Gassen & Schrimpf，Gentechnische Methoden，Spektrum

Akademischer Verlag，Heidelberg，1999

(12)=Malinowski etc., biological chemistry (Biochemistry), 23,1984

(13)=Stack etc., journal of biological chemistry (Biochem.J.) 284,1992

Sequence table

＜110〉N-biotechnology of enzymes company limited

＜120〉method of production recombinant protein in microorganism

<130>P04TK074108

<140>

<141>

<150>EP 02 002 716.5

<151>2002-02-06

<150>US60/357,809

<151>2002-02-21

<160>66

<170>PatentIn Ver.2.1

<210>1

<211>33

<212>DNA

＜213〉homo sapiens (Homo sapiens)

<400>1

aaaaaccgcg gtcaattatt tctcatcact ccc 33

<210>2

<211>37

<212>DNA

＜213〉homo sapiens

<400>2

aaaaactcga gaaaagaaaa gtgtatctct cagagtg 37

<210>3

<211>49

<212>DNA

＜213〉homo sapiens

<400>3

aaaaactcga gaaaagagag gctgaagcta aagtgtatct ctcagagtg 49

<210>4

<211>32

<212>DNA

＜213〉homo sapiens

<400>4

aaaaattcga aaaatggaac ataaggaagt gg 32

<210>5

<211>35

<212>DNA

＜213〉homo sapiens

<400>5

aaaaactcga gaaaagagag cctctggatg actat 35

<210>6

<211>47

<212>DNA

＜213〉homo sapiens

<400>6

aaaaactcga gaaaagagag gctgaagctg agcctctgga tgactat 47

<210>7

<211>2400

<212>DNA

＜213〉homo sapiens

<400>7

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagaaaagt gtatctctca gagtgcaaga ctgggaatgg aaagaactac 300

agagggacga tgtccaaaac aaaaaatggc atcacctgtc aaaaatggag ttccacttct 360

ccccacagac ctagattctc acctgctaca cacccctcag agggactgga ggagaactac 420

tgcaggaatc cagacaacga tccgcagggg ccctggtgct atactactga tccagaaaag 480

agatatgact actgcgacat tcttgagtgt gaagaggaat gtatgcattg cagtggagaa 540

aactatgacg gcaaaatttc caagaccatg tctggactgg aatgccaggc ctgggactct 600

cagagcccac acgctcatgg atacattcct tccaaatttc caaacaagaa cctgaagaag 660

aattactgtc gtaaccccga tagggagctg cggccttggt gtttcaccac cgaccccaac 720

aagcgctggg aactttgcga catcccccgc tgcacaacac ctccaccatc ttctggtccc 780

acctaccagt gtctgaaggg aacaggtgaa aactatcgcg ggaatgtggc tgttaccgtt 840

tccgggcaca cctgtcagca ctggagtgca cagacccctc acacacataa caggacacca 900

gaaaacttcc cctgcaaaaa tttggatgaa aactactgcc gcaatcctga cggaaaaagg 960

gccccatggt gccatacaac caacagccaa gtgcggtggg agtactgtaa gataccgtcc 1020

tgtgactcct ccccagtatc cacggaacaa ttggctccca cagcaccacc tgagctaacc 1080

cctgtggtcc aggactgcta ccatggtgat ggacagagct accgaggcac atcctccacc 1140

accaccacag gaaagaagtg tcagtcttgg tcatctatga caccacaccg gcaccagaag 1200

accccagaaa actacccaaa tgctggcctg acaatgaact actgcaggaa tccagatgcc 1260

gataaaggcc cctggtgttt taccacagac cccagcgtca ggtgggagta ctgcaacctg 1320

aaaaaatgct caggaacaga agcgagtgtt gtagcacctc cgcctgttgt cctgcttcca 1380

gatgtagaga ctccttccga agaagactgt atgtttggga atgggaaagg ataccgaggc 1440

aagagggcga ccactgttac tgggacgcca tgccaggact gggctgccca ggagccccat 1500

agacacagca ttttcactcc agagacaaat ccacgggcgg gtctggaaaa aaattactgc 1560

cgtaaccctg atggtgatgt aggtggtccc tggtgctaca cgacaaatcc aagaaaactt 1620

tacgactact gtgatgtccc tcagtgtgcg gccccttcat ttgattgtgg gaagcctcaa 1680

gtggagccga agaaatgtcc tggaagggtt gtgggggggt gtgtggccca cccacattcc 1740

tggccctggc aagtcagtct tagaacaagg tttggaatgc acttctgtgg aggcaccttg 1800

atatccccag agtgggtgtt gactgctgcc cactgcttgg agaagtcccc aaggccttca 1860

tcctacaagg tcatcctggg tgcacaccaa gaagtgaatc tcgaaccgca tgttcaggaa 1920

atagaagtgt ctaggctgtt cttggagccc acacgaaaag atattgcctt gctaaagcta 1980

agcagtcctg ccgtcatcac tgacaaagta atcccagctt gtctgccatc cccaaattat 2040

gtggtcgctg accggaccga atgtttcatc actggctggg gagaaaccca aggtactttt 2100

ggagctggcc ttctcaagga agcccagctc cctgtgattg agaataaagt gtgcaatcgc 2160

tatgagtttc tgaatggaag agtccaatcc accgaactct gtgctgggca tttggccgga 2220

ggcactgaca gttgccaggg tgacagtgga ggtcctctgg tttgcttcga gaaggacaaa 2280

tacattttac aaggagtcac ttcttggggt cttggctgtg cacgccccaa taagcctggt 2340

gtctatgttc gtgtttcaag gtttgttact tggattgagg gagtgatgag aaataattga 2400

<210>8

<211>799

<212>PRT

＜213〉homo sapiens

<400>8

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn

85 90 95

Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr

100 105 110

Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro

115 120 125

Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro

130 135 140

Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys

145 150 155 160

Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His

165 170 175

Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly

180 185 190

Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr

195 200 205

Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg

210 215 220

Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn

225 230 235 240

Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro

245 250 255

Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr

260 265 270

Arg Gly Asn Val Ala Val Thr Val Ser Gly His Thr Cys Gln His Trp

275 280 285

Ser Ala Gln Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe Pro

290 295 300

Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg

305 310 315 320

Ala Pro Trp Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys

325 330 335

Lys Ile Pro Ser Cys Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala

340 345 350

Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His

355 360 365

Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly

370 375 380

Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Arg His Gln Lys

385 390 395 400

Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg

405 410 415

Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser

420 425 430

Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala

435 440 445

Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr

450 455 460

Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly

465 470 475 480

Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala

485 490 495

Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg

500 505 510

Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly

515 520 525

Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys

530 535 540

Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln

545 550 555 560

Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala

565 570 575

His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly

580 585 590

Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr

595 600 605

Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val

610 615 620

Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu

625 630 635 640

Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala

645 650 655

Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro

660 665 670

Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys

675 680 685

Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu

690 695 700

Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg

705 710 715 720

Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly

725 730 735

His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro

740 745 750

Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser

755 760 765

Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg

770 775 780

Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

785 790 795

<210>9

<211>2412

<212>DNA

＜213〉homo sapiens

<400>9

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctaaa gtgtatctct cagagtgcaa gactgggaat 300

ggaaagaact acagagggac gatgtccaaa acaaaaaatg gcatcacctg tcaaaaatgg 360

agttccactt ctccccacag acctagattc tcacctgcta cacacccctc agagggactg 420

gaggagaact actgcaggaa tccagacaac gatccgcagg ggccctggtg ctatactact 480

gatccagaaa agagatatga ctactgcgac attcttgagt gtgaagagga atgtatgcat 540

tgcagtggag aaaactatga cggcaaaatt tccaagacca tgtctggact ggaatgccag 600

gcctgggact ctcagagccc acacgctcat ggatacattc cttccaaatt tccaaacaag 660

aacctgaaga agaattactg tcgtaacccc gatagggagc tgcggccttg gtgtttcacc 720

accgacccca acaagcgctg ggaactttgc gacatccccc gctgcacaac acctccacca 780

tcttctggtc ccacctacca gtgtctgaag ggaacaggtg aaaactatcg cgggaatgtg 840

gctgttaccg tttccgggca cacctgtcag cactggagtg cacagacccc tcacacacat 900

aacaggacac cagaaaactt cccctgcaaa aatttggatg aaaactactg ccgcaatcct 960

gacggaaaaa gggccccatg gtgccataca accaacagcc aagtgcggtg ggagtactgt 1020

aagataccgt cctgtgactc ctccccagta tccacggaac aattggctcc cacagcacca 1080

cctgagctaa cccctgtggt ccaggactgc taccatggtg atggacagag ctaccgaggc 1140

acatcctcca ccaccaccac aggaaagaag tgtcagtctt ggtcatctat gacaccacac 1200

cggcaccaga agaccccaga aaactaccca aatgctggcc tgacaatgaa ctactgcagg 1260

aatccagatg ccgataaagg cccctggtgt tttaccacag accccagcgt caggtgggag 1320

tactgcaacc tgaaaaaatg ctcaggaaca gaagcgagtg ttgtagcacc tccgcctgtt 1380

gtcctgcttc cagatgtaga gactccttcc gaagaagact gtatgtttgg gaatgggaaa 1440

ggataccgag gcaagagggc gaccactgtt actgggacgc catgccagga ctgggctgcc 1500

caggagcccc atagacacag cattttcact ccagagacaa atccacgggc gggtctggaa 1560

aaaaattact gccgtaaccc tgatggtgat gtaggtggtc cctggtgcta cacgacaaat 1620

ccaagaaaac tttacgacta ctgtgatgtc cctcagtgtg cggccccttc atttgattgt 1680

gggaagcctc aagtggagcc gaagaaatgt cctggaaggg ttgtgggggg gtgtgtggcc 1740

cacccacatt cctggccctg gcaagtcagt cttagaacaa ggtttggaat gcacttctgt 1800

ggaggcacct tgatatcccc agagtgggtg ttgactgctg cccactgctt ggagaagtcc 1860

ccaaggcctt catcctacaa ggtcatcctg ggtgcacacc aagaagtgaa tctcgaaccg 1920

catgttcagg aaatagaagt gtctaggctg ttcttggagc ccacacgaaa agatattgcc 1980

ttgctaaagc taagcagtcc tgccgtcatc actgacaaag taatcccagc ttgtctgcca 2040

tccccaaatt atgtggtcgc tgaccggacc gaatgtttca tcactggctg gggagaaacc 2100

caaggtactt ttggagctgg ccttctcaag gaagcccagc tccctgtgat tgagaataaa 2160

gtgtgcaatc gctatgagtt tctgaatgga agagtccaat ccaccgaact ctgtgctggg 2220

catttggccg gaggcactga cagttgccag ggtgacagtg gaggtcctct ggtttgcttc 2280

gagaaggaca aatacatttt acaaggagtc acttcttggg gtcttggctg tgcacgcccc 2340

aataagcctg gtgtctatgt tcgtgtttca aggtttgtta cttggattga gggagtgatg 2400

agaaataatt ga 2412

<210>10

<211>803

<212>PRT

＜213〉homo sapiens

<400>10

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Lys Val Tyr Leu Ser Glu Cys

85 90 95

Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys

100 105 110

Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro

115 120 125

Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr

130 135 140

Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr

145 150 155 160

Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu

165 170 175

Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys

180 185 190

Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His

195 200 205

Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys

210 215 220

Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr

225 230 235 240

Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr

245 250 255

Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr

260 265 270

Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly His Thr

275 280 285

Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr Pro

290 295 300

Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro

305 310 315 320

Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn Ser Gln Val Arg

325 330 335

Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser Pro Val Ser Thr

340 345 350

Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val Gln

355 360 365

Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr

370 375 380

Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His

385 390 395 400

Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met

405 410 415

Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr

420 425 430

Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser

435 440 445

Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro

450 455 460

Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys

465 470 475 480

Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln

485 490 495

Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu

500 505 510

Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp

515 520 525

Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu

530 535 540

Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys

545 550 555 560

Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly

565 570 575

Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg

580 585 590

Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu

595 600 605

Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser

610 615 620

Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro

625 630 635 640

His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg

645 650 655

Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp

660 665 670

Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp

675 680 685

Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe

690 695 700

Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys

705 710 715 720

Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu

725 730 735

Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp

740 745 750

Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln

755 760 765

Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly

770 775 780

Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met

785 790 795 800

Arg Asn Asn

<210>11

<211>2433

<212>DNA

＜213〉homo sapiens

<400>11

atggaacata aggaagtggt tcttctactt cttttatttc tgaaatcagg tcaaggagag 60

cctctggatg actatgtgaa tacccagggg gcttcactgt tcagtgtcac taagaagcag 120

ctgggagcag gaagtataga agaatgtgca gcaaaatgtg aggaggacga agaattcacc 180

tgcagggcat tccaatatca cagtaaagag caacaatgtg tgataatggc tgaaaacagg 240

aagtcctcca taatcattag gatgagagat gtagttttat ttgaaaagaa agtgtatctc 300

tcagagtgca agactgggaa tggaaagaac tacagaggga cgatgtccaa aacaaaaaat 360

ggcatcacct gtcaaaaatg gagttccact tctccccaca gacctagatt ctcacctgct 420

acacacccct cagagggact ggaggagaac tactgcagga atccagacaa cgatccgcag 480

gggccctggt gctatactac tgatccagaa aagagatatg actactgcga cattcttgag 540

tgtgaagagg aatgtatgca ttgcagtgga gaaaactatg acggcaaaat ttccaagacc 600

atgtctggac tggaatgcca ggcctgggac tctcagagcc cacacgctca tggatacatt 660

ccttccaaat ttccaaacaa gaacctgaag aagaattact gtcgtaaccc cgatagggag 720

ctgcggcctt ggtgtttcac caccgacccc aacaagcgct gggaactttg cgacatcccc 780

cgctgcacaa cacctccacc atcttctggt cccacctacc agtgtctgaa gggaacaggt 840

gaaaactatc gcgggaatgt ggctgttacc gtttccgggc acacctgtca gcactggagt 900

gcacagaccc ctcacacaca taacaggaca ccagaaaact tcccctgcaa aaatttggat 960

gaaaactact gccgcaatcc tgacggaaaa agggccccat ggtgccatac aaccaacagc 1020

caagtgcggt gggagtactg taagataccg tcctgtgact cctccccagt atccacggaa 1080

caattggctc ccacagcacc acctgagcta acccctgtgg tccaggactg ctaccatggt 1140

gatggacaga gctaccgagg cacatcctcc accaccacca caggaaagaa gtgtcagtct 1200

tggtcatcta tgacaccaca ccggcaccag aagaccccag aaaactaccc aaatgctggc 1260

ctgacaatga actactgcag gaatccagat gccgataaag gcccctggtg ttttaccaca 1320

gaccccagcg tcaggtggga gtactgcaac ctgaaaaaat gctcaggaac agaagcgagt 1380

gttgtagcac ctccgcctgt tgtcctgctt ccagatgtag agactccttc cgaagaagac 1440

tgtatgtttg ggaatgggaa aggataccga ggcaagaggg cgaccactgt tactgggacg 1500

ccatgccagg actgggctgc ccaggagccc catagacaca gcattttcac tccagagaca 1560

aatccacggg cgggtctgga aaaaaattac tgccgtaacc ctgatggtga tgtaggtggt 1620

ccctggtgct acacgacaaa tccaagaaaa ctttacgact actgtgatgt ccctcagtgt 1680

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 1740

gttgtggggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 1800

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 1860

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 1920

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 1980

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 2040

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 2100

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 2160

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 2220

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 2280

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 2340

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 2400

acttggattg agggagtgat gagaaataat tga 2433

<210>12

<211>810

<212>PRT

＜213〉homo sapiens

<400>12

Met Glu His Lys Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser

1 5 10 15

Gly Gln Gly Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly Ala Ser

20 25 30

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

35 40 45

Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe

50 55 60

Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg

65 70 75 80

Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys

85 90 95

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

100 105 110

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

115 120 125

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

130 135 140

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

145 150 155 160

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

165 170 175

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

180 185 190

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

195 200 205

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

210 215 220

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

225 230 235 240

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

245 250 255

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

260 265 270

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

275 280 285

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

290 295 300

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

305 310 315 320

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

325 330 335

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

340 345 350

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

355 360 365

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

370 375 380

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

385 390 395 400

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

405 410 415

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

420 425 430

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

435 440 445

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

450 455 460

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

465 470 475 480

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

485 490 495

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

500 505 510

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

515 520 525

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

530 535 540

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

545 550 555 560

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

565 570 575

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

580 585 590

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

595 600 605

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

610 615 620

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

625 630 635 640

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

645 650 655

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

660 665 670

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

675 680 685

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

690 695 700

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

705 710 715 720

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

725 730 735

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

740 745 750

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

755 760 765

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

770 775 780

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

785 790 795 800

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

805 810

<210>13

<211>2631

<212>DNA

＜213〉homo sapiens

<400>13

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagagcc tctggatgac tatgtgaata cccagggggc ttcactgttc 300

agtgtcacta agaagcagct gggagcagga agtatagaag aatgtgcagc aaaatgtgag 360

gaggacgaag aattcacctg cagggcattc caatatcaca gtaaagagca acaatgtgtg 420

ataatggctg aaaacaggaa gtcctccata atcattagga tgagagatgt agttttattt 480

gaaaagaaag tgtatctctc agagtgcaag actgggaatg gaaagaacta cagagggacg 540

atgtccaaaa caaaaaatgg catcacctgt caaaaatgga gttccacttc tccccacaga 600

cctagattct cacctgctac acacccctca gagggactgg aggagaacta ctgcaggaat 660

ccagacaacg atccgcaggg gccctggtgc tatactactg atccagaaaa gagatatgac 720

tactgcgaca ttcttgagtg tgaagaggaa tgtatgcatt gcagtggaga aaactatgac 780

ggcaaaattt ccaagaccat gtctggactg gaatgccagg cctgggactc tcagagccca 840

cacgctcatg gatacattcc ttccaaattt ccaaacaaga acctgaagaa gaattactgt 900

cgtaaccccg atagggagct gcggccttgg tgtttcacca ccgaccccaa caagcgctgg 960

gaactttgcg acatcccccg ctgcacaaca cctccaccat cttctggtcc cacctaccag 1020

tgtctgaagg gaacaggtga aaactatcgc gggaatgtgg ctgttaccgt ttccgggcac 1080

acctgtcagc actggagtgc acagacccct cacacacata acaggacacc agaaaacttc 1140

ccctgcaaaa atttggatga aaactactgc cgcaatcctg acggaaaaag ggccccatgg 1200

tgccatacaa ccaacagcca agtgcggtgg gagtactgta agataccgtc ctgtgactcc 1260

tccccagtat ccacggaaca attggctccc acagcaccac ctgagctaac ccctgtggtc 1320

caggactgct accatggtga tggacagagc taccgaggca catcctccac caccaccaca 1380

ggaaagaagt gtcagtcttg gtcatctatg acaccacacc ggcaccagaa gaccccagaa 1440

aactacccaa atgctggcct gacaatgaac tactgcagga atccagatgc cgataaaggc 1500

ccctggtgtt ttaccacaga ccccagcgtc aggtgggagt actgcaacct gaaaaaatgc 1560

tcaggaacag aagcgagtgt tgtagcacct ccgcctgttg tcctgcttcc agatgtagag 1620

actccttccg aagaagactg tatgtttggg aatgggaaag gataccgagg caagagggcg 1680

accactgtta ctgggacgcc atgccaggac tgggctgccc aggagcccca tagacacagc 1740

attttcactc cagagacaaa tccacgggcg ggtctggaaa aaaattactg ccgtaaccct 1800

gatggtgatg taggtggtcc ctggtgctac acgacaaatc caagaaaact ttacgactac 1860

tgtgatgtcc ctcagtgtgc ggccccttca tttgattgtg ggaagcctca agtggagccg 1920

aagaaatgtc ctggaagggt tgtggggggg tgtgtggccc acccacattc ctggccctgg 1980

caagtcagtc ttagaacaag gtttggaatg cacttctgtg gaggcacctt gatatcccca 2040

gagtgggtgt tgactgctgc ccactgcttg gagaagtccc caaggccttc atcctacaag 2100

gtcatcctgg gtgcacacca agaagtgaat ctcgaaccgc atgttcagga aatagaagtg 2160

tctaggctgt tcttggagcc cacacgaaaa gatattgcct tgctaaagct aagcagtcct 2220

gccgtcatca ctgacaaagt aatcccagct tgtctgccat ccccaaatta tgtggtcgct 2280

gaccggaccg aatgtttcat cactggctgg ggagaaaccc aaggtacttt tggagctggc 2340

cttctcaagg aagcccagct ccctgtgatt gagaataaag tgtgcaatcg ctatgagttt 2400

ctgaatggaa gagtccaatc caccgaactc tgtgctgggc atttggccgg aggcactgac 2460

agttgccagg gtgacagtgg aggtcctctg gtttgcttcg agaaggacaa atacatttta 2520

caaggagtca cttcttgggg tcttggctgt gcacgcccca ataagcctgg tgtctatgtt 2580

cgtgtttcaa ggtttgttac ttggattgag ggagtgatga gaaataattg a 2631

<210>14

<211>876

<212>PRT

＜213〉homo sapiens

<400>14

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Pro Leu Asp Asp Tyr Val Asn Thr Gln Gly

85 90 95

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

100 105 110

Glu Glu Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg

115 120 125

Ala Phe Gln Tyr His Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu

130 135 140

Asn Arg Lys Ser Ser Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe

145 150 155 160

Glu Lys Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn

165 170 175

Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys

180 185 190

Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His

195 200 205

Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp

210 215 220

Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp

225 230 235 240

Tyr Cys Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly

245 250 255

Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys

260 265 270

Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser

275 280 285

Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp

290 295 300

Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp

305 310 315 320

Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly

325 330 335

Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn

340 345 350

Val Ala Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln

355 360 365

Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn

370 375 380

Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp

385 390 395 400

Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro

405 410 415

Ser Cys Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala

420 425 430

Pro Pro Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly

435 440 445

Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys

450 455 460

Gln Ser Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu

465 470 475 480

Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp

485 490 495

Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp

500 505 510

Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val

515 520 525

Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu

530 535 540

Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala

545 550 555 560

Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro

565 570 575

His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu

580 585 590

Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp

595 600 605

Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro

610 615 620

Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro

625 630 635 640

Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His

645 650 655

Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe

660 665 670

Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His

675 680 685

Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly

690 695 700

Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val

705 710 715 720

Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys

725 730 735

Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu

740 745 750

Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr

755 760 765

Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu

770 775 780

Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe

785 790 795 800

Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala

805 810 815

Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys

820 825 830

Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu

835 840 845

Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg

850 855 860

Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

865 870 875

<210>15

<211>2643

<212>DNA

＜213〉homo sapiens

<400>15

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctgag cctctggatg actatgtgaa tacccagggg 300

gcttcactgt tcagtgtcac taagaagcag ctgggagcag gaagtataga agaatgtgca 360

gcaaaatgtg aggaggacga agaattcacc tgcagggcat tccaatatca cagtaaagag 420

caacaatgtg tgataatggc tgaaaacagg aagtcctcca taatcattag gatgagagat 480

gtagttttat ttgaaaagaa agtgtatctc tcagagtgca agactgggaa tggaaagaac 540

tacagaggga cgatgtccaa aacaaaaaat ggcatcacct gtcaaaaatg gagttccact 600

tctccccaca gacctagatt ctcacctgct acacacccct cagagggact ggaggagaac 660

tactgcagga atccagacaa cgatccgcag gggccctggt gctatactac tgatccagaa 720

aagagatatg actactgcga cattcttgag tgtgaagagg aatgtatgca ttgcagtgga 780

gaaaactatg acggcaaaat ttccaagacc atgtctggac tggaatgcca ggcctgggac 840

tctcagagcc cacacgctca tggatacatt ccttccaaat ttccaaacaa gaacctgaag 900

aagaattact gtcgtaaccc cgatagggag ctgcggcctt ggtgtttcac caccgacccc 960

aacaagcgct gggaactttg cgacatcccc cgctgcacaa cacctccacc atcttctggt 1020

cccacctacc agtgtctgaa gggaacaggt gaaaactatc gcgggaatgt ggctgttacc 1080

gtttccgggc acacctgtca gcactggagt gcacagaccc ctcacacaca taacaggaca 1140

ccagaaaact tcccctgcaa aaatttggat gaaaactact gccgcaatcc tgacggaaaa 1200

agggccccat ggtgccatac aaccaacagc caagtgcggt gggagtactg taagataccg 1260

tcctgtgact cctccccagt atccacggaa caattggctc ccacagcacc acctgagcta 1320

acccctgtgg tccaggactg ctaccatggt gatggacaga gctaccgagg cacatcctcc 1380

accaccacca caggaaagaa gtgtcagtct tggtcatcta tgacaccaca ccggcaccag 1440

aagaccccag aaaactaccc aaatgctggc ctgacaatga actactgcag gaatccagat 1500

gccgataaag gcccctggtg ttttaccaca gaccccagcg tcaggtggga gtactgcaac 1560

ctgaaaaaat gctcaggaac agaagcgagt gttgtagcac ctccgcctgt tgtcctgctt 1620

ccagatgtag agactccttc cgaagaagac tgtatgtttg ggaatgggaa aggataccga 1680

ggcaagaggg cgaccactgt tactgggacg ccatgccagg actgggctgc ccaggagccc 1740

catagacaca gcattttcac tccagagaca aatccacggg cgggtctgga aaaaaattac 1800

tgccgtaacc ctgatggtga tgtaggtggt ccctggtgct acacgacaaa tccaagaaaa 1860

ctttacgact actgtgatgt ccctcagtgt gcggcccctt catttgattg tgggaagcct 1920

caagtggagc cgaagaaatg tcctggaagg gttgtggggg ggtgtgtggc ccacccacat 1980

tcctggccct ggcaagtcag tcttagaaca aggtttggaa tgcacttctg tggaggcacc 2040

ttgatatccc cagagtgggt gttgactgct gcccactgct tggagaagtc cccaaggcct 2100

tcatcctaca aggtcatcct gggtgcacac caagaagtga atctcgaacc gcatgttcag 2160

gaaatagaag tgtctaggct gttcttggag cccacacgaa aagatattgc cttgctaaag 2220

ctaagcagtc ctgccgtcat cactgacaaa gtaatcccag cttgtctgcc atccccaaat 2280

tatgtggtcg ctgaccggac cgaatgtttc atcactggct ggggagaaac ccaaggtact 2340

tttggagctg gccttctcaa ggaagcccag ctccctgtga ttgagaataa agtgtgcaat 2400

cgctatgagt ttctgaatgg aagagtccaa tccaccgaac tctgtgctgg gcatttggcc 2460

ggaggcactg acagttgcca gggtgacagt ggaggtcctc tggtttgctt cgagaaggac 2520

aaatacattt tacaaggagt cacttcttgg ggtcttggct gtgcacgccc caataagcct 2580

ggtgtctatg ttcgtgtttc aaggtttgtt acttggattg agggagtgat gagaaataat 2640

tga 2643

<210>16

<211>880

<212>PRT

＜213〉homo sapiens

<400>16

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Ash Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

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

85 90 95

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

100 105 110

Ala Gly Ser Ile Glu Glu Cys Ala Ala Lys Cys Glu Glu Asp Glu Glu

115 120 125

Phe Thr Cys Arg Ala Phe Gln Tyr His Ser Lys Glu Gln Gln Cys Val

130 135 140

Ile Met Ala Glu Asn Arg Lys Ser Ser Ile Ile Ile Arg Met Arg Asp

145 150 155 160

Val Val Leu Phe Glu Lys Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly

165 170 175

Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile

180 185 190

Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser

195 200 205

Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn

210 215 220

Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu

225 230 235 240

Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu Glu Glu Cys Met

245 250 255

His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser

260 265 270

Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly

275 280 285

Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys

290 295 300

Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro

305 310 315 320

Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro

325 330 335

Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn

340 345 350

Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly His Thr Cys Gln His

355 360 365

Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr Pro Glu Asn Phe

370 375 380

Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys

385 390 395 400

Arg Ala Pro Trp Cys His Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr

405 410 415

Cys Lys Ile Pro Ser Cys Asp Ser Ser Pro Val Ser Thr Glu Gln Leu

420 425 430

Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val Gln Asp Cys Tyr

435 440 445

His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr

450 455 460

Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro His Arg His Gln

465 470 475 480

Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys

485 490 495

Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro

500 505 510

Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu

515 520 525

Ala Ser Val Val Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu

530 535 540

Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg

545 550 555 560

Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala

565 570 575

Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro

580 585 590

Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val

595 600 605

Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr

610 615 620

Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro

625 630 635 640

Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val

645 650 655

Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe

660 665 670

Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu

675 680 685

Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys

690 695 700

Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln

705 710 715 720

Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile

725 730 735

Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile

740 745 750

Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu

755 760 765

Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly

770 775 780

Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn

785 790 795 800

Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala

805 810 815

Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly

820 825 830

Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr

835 840 845

Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val

850 855 860

Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

865 870 875 880

<210>17

<211>791

<212>PRT

＜213〉homo sapiens

<400>17

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

1 5 10 15

Val Thr Lys Lys Gln Leu Gly Ala Gly Ser Ile Glu Glu Cys Ala Ala

20 25 30

Lys Cys Glu Glu Asp Glu Glu Phe Thr Cys Arg Ala Phe Gln Tyr His

35 40 45

Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Arg Lys Ser Ser

50 55 60

Ile Ile Ile Arg Met Arg Asp Val Val Leu Phe Glu Lys Lys Val Tyr

65 70 75 80

Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met

85 90 95

Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser

100 105 110

Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu

115 120 125

Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp

130 135 140

Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu

145 150 155 160

Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly

165 170 175

Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser

180 185 190

Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys

195 200 205

Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro

210 215 220

Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile

225 230 235 240

Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys

245 250 255

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

260 265 270

Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His

275 280 285

Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr

290 295 300

Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn

305 310 315 320

Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Asp Ser Ser

325 330 335

Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro Glu Leu Thr

340 345 350

Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly

355 360 365

Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser

370 375 380

Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala

385 390 395 400

Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro

405 410 415

Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu

420 425 430

Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro Pro Pro Val

435 440 445

Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe

450 455 460

Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly

465 470 475 480

Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile

485 490 495

Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys

500 505 510

Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn

515 520 525

Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro

530 535 540

Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly

545 550 555 560

Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln

565 570 575

Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu

580 585 590

Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser

595 600 605

Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val

610 615 620

Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu

625 630 635 640

Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala

645 650 655

Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr

660 665 670

Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr

675 680 685

Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val

690 695 700

Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val

705 710 715 720

Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser

725 730 735

Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys

740 745 750

Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro

755 760 765

Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile

770 775 780

Glu Gly Val Met Arg Asn Asn

785 790

<210>18

<211>714

<212>PRT

＜213〉homo sapiens

<400>18

Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg

1 5 10 15

Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser

20 25 30

Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser

35 40 45

Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln

50 55 60

Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys

65 70 75 80

Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn

85 90 95

Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala

100 105 110

Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe

115 120 125

Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu

130 135 140

Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu

145 150 155 160

Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr

165 170 175

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

180 185 190

Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro

195 200 205

His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp

210 215 220

Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His

225 230 235 240

Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys

245 250 255

Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro

260 265 270

Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser

275 280 285

Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser

290 295 300

Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr

305 310 315 320

Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp

325 330 335

Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr

340 345 350

Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro

355 360 365

Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp

370 375 380

Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr

385 390 395 400

Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg

405 410 415

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

420 425 430

Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr

435 440 445

Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

450 455 460

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

465 470 475 480

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

485 490 495

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

500 505 510

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

515 520 525

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

530 535 540

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

545 550 555 560

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

565 570 575

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

580 585 590

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

595 600 605

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

610 615 620

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

625 630 635 640

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

645 650 655

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

660 665 670

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

675 680 685

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

690 695 700

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

705 710

<210>19

<211>33

<212>DNA

＜213〉homo sapiens

<400>19

aaaaactcga gaaaagagca cctccgcctg ttg 33

<210>20

<211>45

<212>DNA

＜213〉homo sapiens

<400>20

aaaaactcga gaaaagagag gctgaagctg cacctccgcc tgttg 45

<210>2l

<211>34

<212>DNA

＜213〉homo sapiens

<400>21

aaaaactcga gaaaagaaaa ctttacgact actg 34

<210>22

<211>46

<212>DNA

＜213〉homo sapiens

<400>22

aaaaactcga gaaaagagag gctgaagcta aactttacga ctactg 46

<210>23

<211>33

<212>DNA

＜213〉homo sapiens

<400>23

aaaaactcga gaaaagactt tacgactact gtg 33

<210>24

<211>45

<212>DNA

＜213〉homo sapiens

<400>24

aaaaactcga gaaaagagag gctgaagctc tttacgacta ctgtg 45

<210>25

<211>36

<212>DNA

＜213〉homo sapiens

<400>25

aaaaactcga gaaaagagcc ccttcatttg attgtg 36

<210>26

<211>48

<212>DNA

＜213〉homo sapiens

<400>26

aaaaactcga gaaaagagag gctgaagctg ccccttcatt tgattgtg 48

<210>27

<211>37

<212>DNA

＜213〉homo sapiens

<400>27

aaaaactcga gaaaagatca tttgattgtg ggaagcc 37

<210>28

<21l>49

<212>DNA

＜213〉homo sapiens

<400>28

aaaaactcga gaaaagagag gctgaagctt catttgattg tgggaagcc 49

<210>29

<21l>348

<212>PRT

＜213〉homo sapiens

<400>29

Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val Glu Thr Pro Ser Glu

1 5 10 15

Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala

20 25 30

Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp Ala Ala Gln Glu Pro

35 40 45

His Arg His Ser Ile Phe Thr Pro Glu Thr Asn Pro Arg Ala Gly Leu

50 55 60

Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp Val Gly Gly Pro Trp

65 70 75 80

Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro

85 90 95

Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro

100 105 110

Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His

115 120 125

Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe

130 135 140

Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His

145 150 155 160

Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly

165 170 175

Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val

180 185 190

Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys

195 200 205

Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu

210 215 220

Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr

225 230 235 240

Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu

245 250 255

Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe

260 265 270

Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala

275 280 285

Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys

290 295 300

Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu

305 310 315 320

Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg

325 330 335

Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

340 345

<210>30

<211>261

<212>PRT

＜213〉homo sapiens

<400>30

Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe

1 5 10 15

Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val

20 25 30

Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser

35 40 45

Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser

50 55 60

Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg

65 70 75 80

Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu

85 90 95

Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro

100 105 110

Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile

115 120 125

Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val

130 135 140

Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly

145 150 155 160

Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu

165 170 175

Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser

180 185 190

Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln

195 200 205

Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile

210 215 220

Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys

225 230 235 240

Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly

245 250 255

Val Met Arg Asn Asn

260

<210>31

<211>260

<212>PRT

＜213〉homo sapiens

<400>31

Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp

1 5 10 15

Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val

20 25 30

Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu

35 40 45

Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro

50 55 60

Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro

65 70 75 80

Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu

85 90 95

Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr

100 105 110

Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr

115 120 125

Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala

130 135 140

Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr

145 150 155 160

Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn

165 170 175

Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr

180 185 190

Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly

195 200 205

Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu

210 215 220

Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro

225 230 235 240

Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val

245 250 255

Met Arg Asn Asn

260

<210>32

<211>249

<212>PRT

＜213〉homo sapiens

<400>32

Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys

1 5 10 15

Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro

20 25 30

Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly

35 40 45

Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu

50 55 60

Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln

65 70 75 80

Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu

85 90 95

Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser

100 105 110

Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro

115 120 125

Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly

130 135 140

Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu

145 150 155 160

Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly

165 170 175

Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr

180 185 190

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys

195 200 205

Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala

210 215 220

Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr

225 230 235 240

Trp Ile Glu Gly Val Met Arg Asn Asn

245

<210>33

<211>247

<212>PRT

＜213〉homo sapiens

<400>33

Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly

1 5 10 15

Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln

20 25 30

Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu

35 40 45

Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser

50 55 60

Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val

65 70 75 80

Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu

85 90 95

Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala

100 105 110

Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr

115 120 125

Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr

130 135 140

Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val

145 150 155 160

Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val

165 170 175

Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser

180 185 190

Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys

195 200 205

Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro

210 215 220

Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile

225 230 235 240

Glu Gly Val Met Arg Asn Asn

245

<210>34

<211>249

<212>DNA

＜213〉homo sapiens

<400>34

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgag 249

<210>35

<211>83

<212>PRT

＜213〉homo sapiens

<400>35

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu

<210>36

<211>6

<212>DNA

＜213〉homo sapiens

<400>36

aaaaga 6

<210>37

<211>12

<212>DNA

＜213〉homo sapiens

<400>37

gaggctgaag ct 12

<210>38

<211>2

<212>PRT

＜213〉homo sapiens

<400>38

Lys Arg

l

<210>39

<211>4

<212>PRT

＜213〉homo sapiens

<400>39

Glu Ala Glu Ala

1

<210>40

<211>433

<212>PRT

＜213〉homo sapiens

<400>40

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Ala Pro Pro Pro Val Val Leu Leu Pro Asp Val

85 90 95

Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn Gly Lys Gly Tyr

100 105 110

Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys Gln Asp Trp

115 120 125

Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro Glu Thr Asn

130 135 140

Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro Asp Gly Asp

145 150 155 160

Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys Leu Tyr Asp

165 170 175

Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys

180 185 190

Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys

195 200 205

Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg

210 215 220

Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val

225 230 235 240

Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr

245 250 255

Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val

260 265 270

Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp

275 280 285

Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val

290 295 300

Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr

305 310 315 320

Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala

325 330 335

Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys

340 345 350

Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys

355 360 365

Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly

370 375 380

Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val

385 390 395 400

Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr

405 410 415

Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn

420 425 430

Asn

<210>41

<211>437

<212>PRT

＜213〉homo sapiens

<400>41

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Ala Pro Pro Pro Val Val Leu

85 90 95

Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp Cys Met Phe Gly Asn

100 105 110

Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro

115 120 125

Cys Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr

130 135 140

Pro Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn

145 150 155 160

Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg

165 170 175

Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Phe

180 185 190

Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val

195 200 205

Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser

210 215 220

Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser

225 230 235 240

Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg

245 250 255

Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu

260 265 270

Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro

275 280 285

Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile

290 295 300

Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val

305 310 315 320

Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly

325 330 335

Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu

340 345 350

Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser

355 360 365

Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln

370 375 380

Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile

385 390 395 400

Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys

405 410 415

Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly

420 425 430

Val Met Arg Asn Asn

435

<210>42

<211>346

<212>PRT

＜213〉homo sapiens

<400>42

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Lys Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys

85 90 95

Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys

100 105 110

Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp

115 120 125

Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly

130 135 140

Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu

145 150 155 160

Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His

165 170 175

Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg

180 185 190

Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser

195 200 205

Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser

210 215 220

Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp

225 230 235 240

Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln

245 250 255

Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn

260 265 270

Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly

275 280 285

Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu

290 295 300

Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys

305 310 315 320

Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val

325 330 335

Thr Trp Ile Glu Gly Val Met Arg Asn Asn

340 345

<210>43

<211>350

<212>PRT

＜213〉homo sapiens

<400>43

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Lys Leu Tyr Asp Tyr Cys Asp

85 90 95

Val Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val

100 105 110

Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His

115 120 125

Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met

130 135 140

His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala

145 150 155 160

Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile

165 170 175

Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile

180 185 190

Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu

195 200 205

Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala

210 215 220

Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe

225 230 235 240

Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu

245 250 255

Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr

260 265 270

Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His

275 280 285

Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu

290 295 300

Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp

305 310 315 320

Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val

325 330 335

Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

340 345 350

<210>44

<211>345

<212>PRT

＜213〉homo sapiens

<400>44

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala

85 90 95

Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro Lys Lys Cys

100 105 110

Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp Pro

115 120 125

Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe Cys Gly Gly

130 135 140

Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Glu

145 150 155 160

Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly Ala His Gln

165 170 175

Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val Ser Arg Leu

180 185 190

Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys Leu Ser Ser

195 200 205

Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu Pro Ser Pro

210 215 220

Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr Gly Trp Gly

225 230 235 240

Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu Ala Gln Leu

245 250 255

Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe Leu Asn Gly

260 265 270

Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala Gly Gly Thr

275 280 285

Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu Lys

290 295 300

Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu Gly Cys Ala

305 310 315 320

Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val Thr

325 330 335

Trp Ile Glu Gly Val Met Arg Asn Asn

340 345

<210>45

<211>349

<212>PRT

＜213〉homo sapiens

<400>45

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Leu Tyr Asp Tyr Cys Asp Val

85 90 95

Pro Gln Cys Ala Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu

100 105 110

Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro

115 120 125

His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His

130 135 140

Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala

145 150 155 160

His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu

165 170 175

Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu

180 185 190

Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu

195 200 205

Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys

210 215 220

Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile

225 230 235 240

Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys

245 250 255

Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu

260 265 270

Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu

275 280 285

Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val

290 295 300

Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly

305 310 315 320

Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser

325 330 335

Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

340 345

<210>46

<211>334

<212>PRT

＜213〉homo sapiens

<400>46

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Ala Pro Ser Phe Asp Cys Gly Lys Pro Gln Val

85 90 95

Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His

100 105 110

Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met

115 120 125

His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala

130 135 140

Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile

145 150 155 160

Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile

165 170 175

Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu

180 185 190

Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala

195 200 205

Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe

210 215 220

Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu

225 230 235 240

Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr

245 250 255

Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His

260 265 270

Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu

275 280 285

Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp

290 295 300

Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val

305 310 315 320

Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

325 330

<210>47

<211>338

<212>PRT

＜213〉homo sapiens

<400>47

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Ala Pro Ser Phe Asp Cys Gly

85 90 95

Lys Pro Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly

100 105 110

Cys Val Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr

115 120 125

Arg Phe Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp

130 135 140

Val Leu Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser

145 150 155 160

Tyr Lys Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His

165 170 175

Val Gln Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys

180 185 190

Asp Ile Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys

195 200 205

Val Ile Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg

210 215 220

Thr Glu Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly

225 230 235 240

Ala Gly Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val

245 250 255

Cys Asn Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu

260 265 270

Cys Ala Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser

275 280 285

Gly Gly Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly

290 295 300

Val Thr Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val

305 310 315 320

Tyr Val Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg

325 330 335

Asn Asn

<210>48

<21l>332

<212>PRT

＜213〉homo sapiens

<400>48

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Ser Phe Asp Cys Gly Lys Pro Gln Val Glu Pro

85 90 95

Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His

100 105 110

Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe Gly Met His Phe

115 120 125

Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His

130 135 140

Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly

145 150 155 160

Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln Glu Ile Glu Val

165 170 175

Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile Ala Leu Leu Lys

180 185 190

Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile Pro Ala Cys Leu

195 200 205

Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu Cys Phe Ile Thr

210 215 220

Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly Leu Leu Lys Glu

225 230 235 240

Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn Arg Tyr Glu Phe

245 250 255

Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala Gly His Leu Ala

260 265 270

Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys

275 280 285

Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu

290 295 300

Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg

305 310 315 320

Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

325 330

<210>49

<211>336

<212>PRT

＜213〉homo sapiens

<400>49

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Ser Phe Asp Cys Gly Lys Pro

85 90 95

Gln Val Glu Pro Lys Lys Cys Pro Gly Arg Val Val Gly Gly Cys Val

100 105 110

Ala His Pro His Ser Trp Pro Trp Gln Val Ser Leu Arg Thr Arg Phe

115 120 125

Gly Met His Phe Cys Gly Gly Thr Leu Ile Ser Pro Glu Trp Val Leu

130 135 140

Thr Ala Ala His Cys Leu Glu Lys Ser Pro Arg Pro Ser Ser Tyr Lys

145 150 155 160

Val Ile Leu Gly Ala His Gln Glu Val Asn Leu Glu Pro His Val Gln

165 170 175

Glu Ile Glu Val Ser Arg Leu Phe Leu Glu Pro Thr Arg Lys Asp Ile

180 185 190

Ala Leu Leu Lys Leu Ser Ser Pro Ala Val Ile Thr Asp Lys Val Ile

195 200 205

Pro Ala Cys Leu Pro Ser Pro Asn Tyr Val Val Ala Asp Arg Thr Glu

210 215 220

Cys Phe Ile Thr Gly Trp Gly Glu Thr Gln Gly Thr Phe Gly Ala Gly

225 230 235 240

Leu Leu Lys Glu Ala Gln Leu Pro Val Ile Glu Asn Lys Val Cys Asn

245 250 255

Arg Tyr Glu Phe Leu Asn Gly Arg Val Gln Ser Thr Glu Leu Cys Ala

260 265 270

Gly His Leu Ala Gly Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly

275 280 285

Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu Gln Gly Val Thr

290 295 300

Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn Lys Pro Gly Val Tyr Val

305 310 315 320

Arg Val Ser Arg Phe Val Thr Trp Ile Glu Gly Val Met Arg Asn Asn

325 330 335

<210>50

<211>1302

<212>DNA

＜213〉homo sapiens

<400>50

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagcacc tccgcctgtt gtcctgcttc cagatgtaga gactccttcc 300

gaagaagact gtatgtttgg gaatgggaaa ggataccgag gcaagagggc gaccactgtt 360

actgggacgc catgccagga ctgggctgcc caggagcccc atagacacag cattttcact 420

ccagagacaa atccacgggc gggtctggaa aaaaattact gccgtaaccc tgatggtgat 480

gtaggtggtc cctggtgcta cacgacaaat ccaagaaaac tttacgacta ctgtgatgtc 540

cctcagtgtg cggccccttc atttgattgt gggaagcctc aagtggagcc gaagaaatgt 600

cctggaaggg ttgtgggggg gtgtgtggcc cacccacatt cctggccctg gcaagtcagt 660

cttagaacaa ggtttggaat gcacttctgt ggaggcacct tgatatcccc agagtgggtg 720

ttgactgctg cccactgctt ggagaagtcc ccaaggcctt catcctacaa ggtcatcctg 780

ggtgcacacc aagaagtgaa tctcgaaccg catgttcagg aaatagaagt gtctaggctg 840

ttcttggagc ccacacgaaa agatattgcc ttgctaaagc taagcagtcc tgccgtcatc 900

actgacaaag taatcccagc ttgtctgcca tccccaaatt atgtggtcgc tgaccggacc 960

gaatgtttca tcactggctg gggagaaacc caaggtactt ttggagctgg ccttctcaag 1020

gaagcccagc tccctgtgat tgagaataaa gtgtgcaatc gctatgagtt tctgaatgga 1080

agagtccaat ccaccgaact ctgtgctggg catttggccg gaggcactga cagttgccag 1140

ggtgacagtg gaggtcctct ggtttgcttc gagaaggaca aatacatttt acaaggagtc 1200

acttcttggg gtcttggctg tgcacgcccc aataagcctg gtgtctatgt tcgtgtttca 1260

aggtttgtta cttggattga gggagtgatg agaaataatt ga 1302

<210>51

<211>1314

<212>DNA

＜213〉homo sapiens

<400>51

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctgca cctccgcctg ttgtcctgct tccagatgta 300

gagactcctt ccgaagaaga ctgtatgttt gggaatggga aaggataccg aggcaagagg 360

gcgaccactg ttactgggac gccatgccag gactgggctg cccaggagcc ccatagacac 420

agcattttca ctccagagac aaatccacgg gcgggtctgg aaaaaaatta ctgccgtaac 480

cctgatggtg atgtaggtgg tccctggtgc tacacgacaa atccaagaaa actttacgac 540

tactgtgatg tccctcagtg tgcggcccct tcatttgatt gtgggaagcc tcaagtggag 600

ccgaagaaat gtcctggaag ggttgtgggg gggtgtgtgg cccacccaca ttcctggccc 660

tggcaagtca gtcttagaac aaggtttgga atgcacttct gtggaggcac cttgatatcc 720

ccagagtggg tgttgactgc tgcccactgc ttggagaagt ccccaaggcc ttcatcctac 780

aaggtcatcc tgggtgcaca ccaagaagtg aatctcgaac cgcatgttca ggaaatagaa 840

gtgtctaggc tgttcttgga gcccacacga aaagatattg ccttgctaaa gctaagcagt 900

cctgccgtca tcactgacaa agtaatccca gcttgtctgc catccccaaa ttatgtggtc 960

gctgaccgga ccgaatgttt catcactggc tggggagaaa cccaaggtac ttttggagct 1020

ggccttctca aggaagccca gctccctgtg attgagaata aagtgtgcaa tcgctatgag 1080

tttctgaatg gaagagtcca atccaccgaa ctctgtgctg ggcatttggc cggaggcact 1140

gacagttgcc agggtgacag tggaggtcct ctggtttgct tcgagaagga caaatacatt 1200

ttacaaggag tcacttcttg gggtcttggc tgtgcacgcc ccaataagcc tggtgtctat 1260

gttcgtgttt caaggtttgt tacttggatt gagggagtga tgagaaataa ttga 1314

<210>52

<211>1041

<212>DNA

＜213〉homo sapiens

<400>52

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagaaaact ttacgactac tgtgatgtcc ctcagtgtgc ggccccttca 300

tttgattgtg ggaagcctca agtggagccg aagaaatgtc ctggaagggt tgtggggggg 360

tgtgtggccc acccacattc ctggccctgg caagtcagtc ttagaacaag gtttggaatg 420

cacttctgtg gaggcacctt gatatcccca gagtgggtgt tgactgctgc ccactgcttg 480

gagaagtccc caaggccttc atcctacaag gtcatcctgg gtgcacacca agaagtgaat 540

ctcgaaccgc atgttcagga aatagaagtg tctaggctgt tcttggagcc cacacgaaaa 600

gatattgcct tgctaaagct aagcagtcct gccgtcatca ctgacaaagt aatcccagct 660

tgtctgccat ccccaaatta tgtggtcgct gaccggaccg aatgtttcat cactggctgg 720

ggagaaaccc aaggtacttt tggagctggc cttctcaagg aagcccagct ccctgtgatt 780

gagaataaag tgtgcaatcg ctatgagttt ctgaatggaa gagtccaatc caccgaactc 840

tgtgctgggc atttggccgg aggcactgac agttgccagg gtgacagtgg aggtcctctg 900

gtttgcttcg agaaggacaa atacatttta caaggagtca cttcttgggg tcttggctgt 960

gcacgcccca ataagcctgg tgtctatgtt cgtgtttcaa ggtttgttac ttggattgag 1020

ggagtgatga gaaataattg a 1041

<210>53

<211>1053

<212>DNA

＜213〉homo sapiens

<400>53

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctaaa ctttacgact actgtgatgt ccctcagtgt 300

gcggcccctt catttgattg tgggaagcct caagtggagc cgaagaaatg tcctggaagg 360

gttgtggggg ggtgtgtggc ccacccacat tcctggccct ggcaagtcag tcttagaaca 420

aggtttggaa tgcacttctg tggaggcacc ttgatatccc cagagtgggt gttgactgct 480

gcccactgct tggagaagtc cccaaggcct tcatcctaca aggtcatcct gggtgcacac 540

caagaagtga atctcgaacc gcatgttcag gaaatagaag tgtctaggct gttcttggag 600

cccacacgaa aagatattgc cttgctaaag ctaagcagtc ctgccgtcat cactgacaaa 660

gtaatcccag cttgtctgcc atccccaaat tatgtggtcg ctgaccggac cgaatgtttc 720

atcactggct ggggagaaac ccaaggtact tttggagctg gccttctcaa ggaagcccag 780

ctccctgtga ttgagaataa agtgtgcaat cgctatgagt ttctgaatgg aagagtccaa 840

tccaccgaac tctgtgctgg gcatttggcc ggaggcactg acagttgcca gggtgacagt 900

ggaggtcctc tggtttgctt cgagaaggac aaatacattt tacaaggagt cacttcttgg 960

ggtcttggct gtgcacgccc caataagcct ggtgtctatg ttcgtgtttc aaggtttgtt 1020

acttggattg agggagtgat gagaaataat tga 1053

<210>54

<211>1038

<212>DNA

＜213〉homo sapiens

<400>54

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagacttta cgactactgt gatgtccctc agtgtgcggc cccttcattt 300

gattgtggga agcctcaagt ggagccgaag aaatgtcctg gaagggttgt gggggggtgt 360

gtggcccacc cacattcctg gccctggcaa gtcagtctta gaacaaggtt tggaatgcac 420

ttctgtggag gcaccttgat atccccagag tgggtgttga ctgctgccca ctgcttggag 480

aagtccccaa ggccttcatc ctacaaggtc atcctgggtg cacaccaaga agtgaatctc 540

gaaccgcatg ttcaggaaat agaagtgtct aggctgttct tggagcccac acgaaaagat 600

attgccttgc taaagctaag cagtcctgcc gtcatcactg acaaagtaat cccagcttgt 660

ctgccatccc caaattatgt ggtcgctgac cggaccgaat gtttcatcac tggctgggga 720

gaaacccaag gtacttttgg agctggcctt ctcaaggaag cccagctccc tgtgattgag 780

aataaagtgt gcaatcgcta tgagtttctg aatggaagag tccaatccac cgaactctgt 840

gctgggcatt tggccggagg cactgacagt tgccagggtg acagtggagg tcctctggtt 900

tgcttcgaga aggacaaata cattttacaa ggagtcactt cttggggtct tggctgtgca 960

cgccccaata agcctggtgt ctatgttcgt gtttcaaggt ttgttacttg gattgaggga 1020

gtgatgagaa ataattga 1038

<210>55

<211>1050

<212>DNA

＜213〉homo sapiens

<400>55

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctctt tacgactact gtgatgtccc tcagtgtgcg 300

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 360

gtgggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 420

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 480

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 540

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 600

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 660

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 720

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 780

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 840

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 900

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 960

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 1020

tggattgagg gagtgatgag aaataattga 1050

<210>56

<211>1005

<212>DNA

＜213〉homo sapiens

<400>56

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 300

tgtcctggaa gggttgtggg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 360

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 420

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 480

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 540

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 600

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 660

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 720

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 780

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 840

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 900

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 960

tcaaggtttg ttacttggat tgagggagtg atgagaaata attga 1005

<210>57

<211>1017

<212>DNA

＜213〉homo sapiens

<400>57

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagctgcc ccttcatttg attgtgggaa gcctcaagtg 300

gagccgaaga aatgtcctgg aagggttgtg ggggggtgtg tggcccaccc acattcctgg 360

ccctggcaag tcagtcttag aacaaggttt ggaatgcact tctgtggagg caccttgata 420

tccccagagt gggtgttgac tgctgcccac tgcttggaga agtccccaag gccttcatcc 480

tacaaggtca tcctgggtgc acaccaagaa gtgaatctcg aaccgcatgt tcaggaaata 540

gaagtgtcta ggctgttctt ggagcccaca cgaaaagata ttgccttgct aaagctaagc 600

agtcctgccg tcatcactga caaagtaatc ccagcttgtc tgccatcccc aaattatgtg 660

gtcgctgacc ggaccgaatg tttcatcact ggctggggag aaacccaagg tacttttgga 720

gctggccttc tcaaggaagc ccagctccct gtgattgaga ataaagtgtg caatcgctat 780

gagtttctga atggaagagt ccaatccacc gaactctgtg ctgggcattt ggccggaggc 840

actgacagtt gccagggtga cagtggaggt cctctggttt gcttcgagaa ggacaaatac 900

attttacaag gagtcacttc ttggggtctt ggctgtgcac gccccaataa gcctggtgtc 960

tatgttcgtg tttcaaggtt tgttacttgg attgagggag tgatgagaaa taattga 1017

<210>58

<211>999

<212>DNA

＜213〉homo sapiens

<400>58

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagatcatt tgattgtggg aagcctcaag tggagccgaa gaaatgtcct 300

ggaagggttg tgggggggtg tgtggcccac ccacattcct ggccctggca agtcagtctt 360

agaacaaggt ttggaatgca cttctgtgga ggcaccttga tatccccaga gtgggtgttg 420

actgctgccc actgcttgga gaagtcccca aggccttcat cctacaaggt catcctgggt 480

gcacaccaag aagtgaatct cgaaccgcat gttcaggaaa tagaagtgtc taggctgttc 540

ttggagccca cacgaaaaga tattgccttg ctaaagctaa gcagtcctgc cgtcatcact 600

gacaaagtaa tcccagcttg tctgccatcc ccaaattatg tggtcgctga ccggaccgaa 660

tgtttcatca ctggctgggg agaaacccaa ggtacttttg gagctggcct tctcaaggaa 720

gcccagctcc ctgtgattga gaataaagtg tgcaatcgct atgagtttct gaatggaaga 780

gtccaatcca ccgaactctg tgctgggcat ttggccggag gcactgacag ttgccagggt 840

gacagtggag gtcctctggt ttgcttcgag aaggacaaat acattttaca aggagtcact 900

tcttggggtc ttggctgtgc acgccccaat aagcctggtg tctatgttcg tgtttcaagg 960

tttgttactt ggattgaggg agtgatgaga aataattga 999

<210>59

<211>1011

<212>DNA

＜213〉homo sapiens

<400>59

atgagatttc cttcaatttt tactgctgtt ttattcgcag catcctccgc attagctgct 60

ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt 120

tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat 180

aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta 240

tctctcgaga aaagagaggc tgaagcttca tttgattgtg ggaagcctca agtggagccg 300

aagaaatgtc ctggaagggt tgtggggggg tgtgtggccc acccacattc ctggccctgg 360

caagtcagtc ttagaacaag gtttggaatg cacttctgtg gaggcacctt gatatcccca 420

gagtgggtgt tgactgctgc ccactgcttg gagaagtccc caaggccttc atcctacaag 480

gtcatcctgg gtgcacacca agaagtgaat ctcgaaccgc atgttcagga aatagaagtg 540

tctaggctgt tcttggagcc cacacgaaaa gatattgcct tgctaaagct aagcagtcct 600

gccgtcatca ctgacaaagt aatcccagct tgtctgccat ccccaaatta tgtggtcgct 660

gaccggaccg aatgtttcat cactggctgg ggagaaaccc aaggtacttt tggagctggc 720

cttctcaagg aagcccagct ccctgtgatt gagaataaag tgtgcaatcg ctatgagttt 780

ctgaatggaa gagtccaatc caccgaactc tgtgctgggc atttggccgg aggcactgac 840

agttgccagg gtgacagtgg aggtcctctg gtttgcttcg agaaggacaa atacatttta 900

caaggagtca cttcttgggg tcttggctgt gcacgcccca ataagcctgg tgtctatgtt 960

cgtgtttcaa ggtttgttac ttggattgag ggagtgatga gaaataattg a 1011

<210>60

<211>1047

<212>DNA

＜213〉homo sapiens

<400>60

gcacctccgc ctgttgtcct gcttccagat gtagagactc cttccgaaga agactgtatg 60

tttgggaatg ggaaaggata ccgaggcaag agggcgacca ctgttactgg gacgccatgc 120

caggactggg ctgcccagga gccccataga cacagcattt tcactccaga gacaaatcca 180

cgggcgggtc tggaaaaaaa ttactgccgt aaccctgatg gtgatgtagg tggtccctgg 240

tgctacacga caaatccaag aaaactttac gactactgtg atgtccctca gtgtgcggcc 300

ccttcatttg attgtgggaa gcctcaagtg gagccgaaga aatgtcctgg aagggttgtg 360

ggggggtgtg tggcccaccc acattcctgg ccctggcaag tcagtcttag aacaaggttt 420

ggaatgcact tctgtggagg caccttgata tccccagagt gggtgttgac tgctgcccac 480

tgcttggaga agtccccaag gccttcatcc tacaaggtca tcctgggtgc acaccaagaa 540

gtgaatctcg aaccgcatgt tcaggaaata gaagtgtcta ggctgttctt ggagcccaca 600

cgaaaagata ttgccttgct aaagctaagc agtcctgccg tcatcactga caaagtaatc 660

ccagcttgtc tgccatcccc aaattatgtg gtcgctgacc ggaccgaatg tttcatcact 720

ggctggggag aaacccaagg tacttttgga gctggccttc tcaaggaagc ccagctccct 780

gtgattgaga ataaagtgtg caatcgctat gagtttctga atggaagagt ccaatccacc 840

gaactctgtg ctgggcattt ggccggaggc actgacagtt gccagggtga cagtggaggt 900

cctctggttt gcttcgagaa ggacaaatac attttacaag gagtcacttc ttggggtctt 960

ggctgtgcac gccccaataa gcctggtgtc tatgttcgtg tttcaaggtt tgttacttgg 1020

attgagggag tgatgagaaa taattga 1047

<210>61

<211>786

<212>DNA

＜213〉homo sapiens

<400>61

aaactttacg actactgtga tgtccctcag tgtgcggccc cttcatttga ttgtgggaag 60

cctcaagtgg agccgaagaa atgtcctgga agggttgtgg gggggtgtgt ggcccaccca 120

cattcctggc cctggcaagt cagtcttaga acaaggtttg gaatgcactt ctgtggaggc 180

accttgatat ccccagagtg ggtgttgact gctgcccact gcttggagaa gtccccaagg 240

ccttcatcct acaaggtcat cctgggtgca caccaagaag tgaatctcga accgcatgtt 300

caggaaatag aagtgtctag gctgttcttg gagcccacac gaaaagatat tgccttgcta 360

aagctaagca gtcctgccgt catcactgac aaagtaatcc cagcttgtct gccatcccca 420

aattatgtgg tcgctgaccg gaccgaatgt ttcatcactg gctggggaga aacccaaggt 480

acttttggag ctggccttct caaggaagcc cagctccctg tgattgagaa taaagtgtgc 540

aatcgctatg agtttctgaa tggaagagtc caatccaccg aactctgtgc tgggcatttg 600

gccggaggca ctgacagttg ccagggtgac agtggaggtc ctctggtttg cttcgagaag 660

gacaaataca ttttacaagg agtcacttct tggggtcttg gctgtgcacg ccccaataag 720

cctggtgtct atgttcgtgt ttcaaggttt gttacttgga ttgagggagt gatgagaaat 780

aattga 786

<210>62

<211>783

<212>DNA

＜213〉homo sapiens

<400>62

ctttacgact actgtgatgt ccctcagtgt gcggcccctt catttgattg tgggaagcct 60

caagtggagc cgaagaaatg tcctggaagg gttgtggggg ggtgtgtggc ccacccacat 120

tcctggccct ggcaagtcag tcttagaaca aggtttggaa tgcacttctg tggaggcacc 180

ttgatatccc cagagtgggt gttgactgct gcccactgct tggagaagtc cccaaggcct 240

tcatcctaca aggtcatcct gggtgcacac caagaagtga atctcgaacc gcatgttcag 300

gaaatagaag tgtctaggct gttcttggag cccacacgaa aagatattgc cttgctaaag 360

ctaagcagtc ctgccgtcat cactgacaaa gtaatcccag cttgtctgcc atccccaaat 420

tatgtggtcg ctgaccggac cgaatgtttc atcactggct ggggagaaac ccaaggtact 480

tttggagctg gccttctcaa ggaagcccag ctccctgtga ttgagaataa agtgtgcaat 540

cgctatgagt ttctgaatgg aagagtccaa tccaccgaac tctgtgctgg gcatttggcc 600

ggaggcactg acagttgcca gggtgacagt ggaggtcctc tggtttgctt cgagaaggac 660

aaatacattt tacaaggagt cacttcttgg ggtcttggct gtgcacgccc caataagcct 720

ggtgtctatg ttcgtgtttc aaggtttgtt acttggattg agggagtgat gagaaataat 780

tga 783

<210>63

<211>750

<212>DNA

＜213〉homo sapiens

<400>63

gccccttcat ttgattgtgg gaagcctcaa gtggagccga agaaatgtcc tggaagggtt 60

gtgggggggt gtgtggccca cccacattcc tggccctggc aagtcagtct tagaacaagg 120

tttggaatgc acttctgtgg aggcaccttg atatccccag agtgggtgtt gactgctgcc 180

cactgcttgg agaagtcccc aaggccttca tcctacaagg tcatcctggg tgcacaccaa 240

gaagtgaatc tcgaaccgca tgttcaggaa atagaagtgt ctaggctgtt cttggagccc 300

acacgaaaag atattgcctt gctaaagcta agcagtcctg ccgtcatcac tgacaaagta 360

atcccagctt gtctgccatc cccaaattat gtggtcgctg accggaccga atgtttcatc 420

actggctggg gagaaaccca aggtactttt ggagctggcc ttctcaagga agcccagctc 480

cctgtgattg agaataaagt gtgcaatcgc tatgagtttc tgaatggaag agtccaatcc 540

accgaactct gtgctgggca tttggccgga ggcactgaca gttgccaggg tgacagtgga 600

ggtcctctgg tttgcttcga gaaggacaaa tacattttac aaggagtcac ttcttggggt 660

cttggctgtg cacgccccaa taagcctggt gtctatgttc gtgtttcaag gtttgttact 720

tggattgagg gagtgatgag aaataattga 750

<210>64

<211>744

<212>DNA

＜213〉homo sapiens

<400>64

tcatttgatt gtgggaagcc tcaagtggag ccgaagaaat gtcctggaag ggttgtgggg 60

gggtgtgtgg cccacccaca ttcctggccc tggcaagtca gtcttagaac aaggtttgga 120

atgcacttct gtggaggcac cttgatatcc ccagagtggg tgttgactgc tgcccactgc 180

ttggagaagt ccccaaggcc ttcatcctac aaggtcatcc tgggtgcaca ccaagaagtg 240

aatctcgaac cgcatgttca ggaaatagaa gtgtctaggc tgttcttgga gcccacacga 300

aaagatattg ccttgctaaa gctaagcagt cctgccgtca tcactgacaa agtaatccca 360

gcttgtctgc catccccaaa ttatgtggtc gctgaccgga ccgaatgttt catcactggc 420

tggggagaaa cccaaggtac ttttggagct ggccttctca aggaagccca gctccctgtg 480

attgagaata aagtgtgcaa tcgctatgag tttctgaatg gaagagtcca atccaccgaa 540

ctctgtgctg ggcatttggc cggaggcact gacagttgcc agggtgacag tggaggtcct 600

ctggtttgct tcgagaagga caaatacatt ttacaaggag tcacttcttg gggtcttggc 660

tgtgcacgcc ccaataagcc tggtgtctat gttcgtgttt caaggtttgt tacttggatt 720

gagggagtga tgagaaataa ttga 744

<210>65

<211>2376

<212>DNA

＜213〉homo sapiens

<400>65

gagcctctgg atgactatgt gaatacccag ggggcttcac tgttcagtgt cactaagaag 60

cagctgggag caggaagtat agaagaatgt gcagcaaaat gtgaggagga cgaagaattc 120

acctgcaggg cattccaata tcacagtaaa gagcaacaat gtgtgataat ggctgaaaac 180

aggaagtcct ccataatcat taggatgaga gatgtagttt tatttgaaaa gaaagtgtat 240

ctctcagagt gcaagactgg gaatggaaag aactacagag ggacgatgtc caaaacaaaa 300

aatggcatca cctgtcaaaa atggagttcc acttctcccc acagacctag attctcacct 360

gctacacacc cctcagaggg actggaggag aactactgca ggaatccaga caacgatccg 420

caggggccct ggtgctatac tactgatcca gaaaagagat atgactactg cgacattctt 480

gagtgtgaag aggaatgtat gcattgcagt ggagaaaact atgacggcaa aatttccaag 540

accatgtctg gactggaatg ccaggcctgg gactctcaga gcccacacgc tcatggatac 600

attccttcca aatttccaaa caagaacctg aagaagaatt actgtcgtaa ccccgatagg 660

gagctgcggc cttggtgttt caccaccgac cccaacaagc gctgggaact ttgcgacatc 720

ccccgctgca caacacctcc accatcttct ggtcccacct accagtgtct gaagggaaca 780

ggtgaaaact atcgcgggaa tgtggctgtt accgtttccg ggcacacctg tcagcactgg 840

agtgcacaga cccctcacac acataacagg acaccagaaa acttcccctg caaaaatttg 900

gatgaaaact actgccgcaa tcctgacgga aaaagggccc catggtgcca tacaaccaac 960

agccaagtgc ggtgggagta ctgtaagata ccgtcctgtg actcctcccc agtatccacg 1020

gaacaattgg ctcccacagc accacctgag ctaacccctg tggtccagga ctgctaccat 1080

ggtgatggac agagctaccg aggcacatcc tccaccacca ccacaggaaa gaagtgtcag 1140

tcttggtcat ctatgacacc acaccggcac cagaagaccc cagaaaacta cccaaatgct 1200

ggcctgacaa tgaactactg caggaatcca gatgccgata aaggcccctg gtgttttacc 1260

acagacccca gcgtcaggtg ggagtactgc aacctgaaaa aatgctcagg aacagaagcg 1320

agtgttgtag cacctccgcc tgttgtcctg cttccagatg tagagactcc ttccgaagaa 1380

gactgtatgt ttgggaatgg gaaaggatac cgaggcaaga gggcgaccac tgttactggg 1440

acgccatgcc aggactgggc tgcccaggag ccccatagac acagcatttt cactccagag 1500

acaaatccac gggcgggtct ggaaaaaaat tactgccgta accctgatgg tgatgtaggt 1560

ggtccctggt gctacacgac aaatccaaga aaactttacg actactgtga tgtccctcag 1620

tgtgcggccc cttcatttga ttgtgggaag cctcaagtgg agccgaagaa atgtcctgga 1680

agggttgtgg gggggtgtgt ggcccaccca cattcctggc cctggcaagt cagtcttaga 1740

acaaggtttg gaatgcactt ctgtggaggc accttgatat ccccagagtg ggtgttgact 1800

gctgcccact gcttggagaa gtccccaagg ccttcatcct acaaggtcat cctgggtgca 1860

caccaagaag tgaatctcga accgcatgtt caggaaatag aagtgtctag gctgttcttg 1920

gagcccacac gaaaagatat tgccttgcta aagctaagca gtcctgccgt catcactgac 1980

aaagtaatcc cagcttgtct gccatcccca aattatgtgg tcgctgaccg gaccgaatgt 2040

ttcatcactg gctggggaga aacccaaggt acttttggag ctggccttct caaggaagcc 2100

cagctccctg tgattgagaa taaagtgtgc aatcgctatg agtttctgaa tggaagagtc 2160

caatccaccg aactctgtgc tgggcatttg gccggaggca ctgacagttg ccagggtgac 2220

agtggaggtc ctctggtttg cttcgagaag gacaaataca ttttacaagg agtcacttct 2280

tggggtcttg gctgtgcacg ccccaataag cctggtgtct atgttcgtgt ttcaaggttt 2340

gttacttgga ttgagggagt gatgagaaat aattga 2376

<210>66

<211>2145

<212>DNA

＜213〉homo sapiens

<400>66

aaagtgtatc tctcagagtg caagactggg aatggaaaga actacagagg gacgatgtcc 60

aaaacaaaaa atggcatcac ctgtcaaaaa tggagttcca cttctcccca cagacctaga 120

ttctcacctg ctacacaccc ctcagaggga ctggaggaga actactgcag gaatccagac 180

aacgatccgc aggggccctg gtgctatact actgatccag aaaagagata tgactactgc 240

gacattcttg agtgtgaaga ggaatgtatg cattgcagtg gagaaaacta tgacggcaaa 300

atttccaaga ccatgtctgg actggaatgc caggcctggg actctcagag cccacacgct 360

catggataca ttccttccaa atttccaaac aagaacctga agaagaatta ctgtcgtaac 420

cccgataggg agctgcggcc ttggtgtttc accaccgacc ccaacaagcg ctgggaactt 480

tgcgacatcc cccgctgcac aacacctcca ccatcttctg gtcccaccta ccagtgtctg 540

aagggaacag gtgaaaacta tcgcgggaat gtggctgtta ccgtttccgg gcacacctgt 600

cagcactgga gtgcacagac ccctcacaca cataacagga caccagaaaa cttcccctgc 660

aaaaatttgg atgaaaacta ctgccgcaat cctgacggaa aaagggcccc atggtgccat 720

acaaccaaca gccaagtgcg gtgggagtac tgtaagatac cgtcctgtga ctcctcccca 780

gtatccacgg aacaattggc tcccacagca ccacctgagc taacccctgt ggtccaggac 840

tgctaccatg gtgatggaca gagctaccga ggcacatcct ccaccaccac cacaggaaag 900

aagtgtcagt cttggtcatc tatgacacca caccggcacc agaagacccc agaaaactac 960

ccaaatgctg gcctgacaat gaactactgc aggaatccag atgccgataa aggcccctgg 1020

tgttttacca cagaccccag cgtcaggtgg gagtactgca acctgaaaaa atgctcagga 1080

acagaagcga gtgttgtagc acctccgcct gttgtcctgc ttccagatgt agagactcct 1140

tccgaagaag actgtatgtt tgggaatggg aaaggatacc gaggcaagag ggcgaccact 1200

gttactggga cgccatgcca ggactgggct gcccaggagc cccatagaca cagcattttc 1260

actccagaga caaatccacg ggcgggtctg gaaaaaaatt actgccgtaa ccctgatggt 1320

gatgtaggtg gtccctggtg ctacacgaca aatccaagaa aactttacga ctactgtgat 1380

gtccctcagt gtgcggcccc ttcatttgat tgtgggaagc ctcaagtgga gccgaagaaa 1440

tgtcctggaa gggttgtggg ggggtgtgtg gcccacccac attcctggcc ctggcaagtc 1500

agtcttagaa caaggtttgg aatgcacttc tgtggaggca ccttgatatc cccagagtgg 1560

gtgttgactg ctgcccactg cttggagaag tccccaaggc cttcatccta caaggtcatc 1620

ctgggtgcac accaagaagt gaatctcgaa ccgcatgttc aggaaataga agtgtctagg 1680

ctgttcttgg agcccacacg aaaagatatt gccttgctaa agctaagcag tcctgccgtc 1740

atcactgaca aagtaatccc agcttgtctg ccatccccaa attatgtggt cgctgaccgg 1800

accgaatgtt tcatcactgg ctggggagaa acccaaggta cttttggagc tggccttctc 1860

aaggaagccc agctccctgt gattgagaat aaagtgtgca atcgctatga gtttctgaat 1920

ggaagagtcc aatccaccga actctgtgct gggcatttgg ccggaggcac tgacagttgc 1980

cagggtgaca gtggaggtcc tctggtttgc ttcgagaagg acaaatacat tttacaagga 2040

gtcacttctt ggggtcttgg ctgtgcacgc cccaataagc ctggtgtcta tgttcgtgtt 2100

tcaaggtttg ttacttggat tgagggagtg atgagaaata attga 2145

Claims

1, a kind of in microorganism the method for the functional Profibrinolysin of recombinant production, described method may further comprise the steps at least:

A) will encode at least nucleotide sequence of nucleotide sequence and the segment signal peptide of encoding at least of funtion part of Profibrinolysin merges, for cleavable signal peptide, the nucleotide sequence of described encoding function Profibrinolysin and at least the nucleotide sequence of coded signal peptide by the restriction enzyme site codon coupling of proteolytic enzyme.

2, recombinant method for production as claimed in claim 1, wherein, the nucleotide sequence of the funtion part of the described Profibrinolysin of encoding at least comprises the proteolysis structural domain of Profibrinolysin or its varient or fragment.

3, recombinant method for production as claimed in claim 1 or 2, wherein, the nucleic acid sequence encoding L-glutamic acid Profibrinolysin of the funtion part of the described Profibrinolysin of encoding at least or Methionin Profibrinolysin.

4, as each described recombinant method for production of claim 1 to 3, wherein, the nucleic acid sequence encoding propetide of the described segment signal peptide of encoding at least is former, propetide or former peptide; And/or the restriction enzyme site of described codon proteins encoded enzyme Kex2 and/or Ste13.

5, recombinant method for production as claimed in claim 4 is characterized in that, the signal peptide of the nucleic acid sequence encoding alpha factor of at least one segment signal peptide of described coding, or the signal peptide of the SUC2 of yeast saccharomyces cerevisiae, PHA-E or PHO1.

6, the described recombinant method for production of each claim as described above is characterized in that, produces the functional L-glutamic acid Profibrinolysin or the Methionin Profibrinolysin that are at least 20mg/l.

7, the described recombinant method for production of each claim as described above is characterized in that, after through 120 hours processing, produces the functional Methionin Profibrinolysin that is at least 120U/l.

8, the described recombinant method for production of each claim as described above, wherein, two Oligonucleolide primers that are used to increase are selected from: NO36a (Seq.ID No.19), NO36b (Seq.ID No.20), NO36c (Seq.ID No.21), NO36d (Seq.ID No.22), NO36e (Seq.ID No.23), NO36f (Seq.ID No.24), NO36g (Seq.ID No.25), NO36h (Seq.ID No.26), NO36i (Seq.ID No.27) or NO36j (Seq.ID No.28).

9, the described recombinant method for production of each claim as described above, wherein, two Oligonucleolide primers that are used to increase are selected from: NO34 (Seq.ID No.1), NO36 (Seq.ID NO.2), NO57 (Seq.ID No.3), NO37 (Seq.ID No.4), NO35 (Seq.ID No.5), NO56 (Seq.ID No.6); And be selected from: NO36a (Seq.ID No.19), NO36b (Seq.ID No.20), NO36c (Seq.ID No.21), NO36d (Seq.ID No.22), NO36e (Seq.ID No.23), NO36f (Seq.ID No.24), NO36g (Seq.ID No.25), NO36h (Seq.ID No.26), NO36i (Seq.ID No.27) or NO36j (Seq.ID No.28).

10, the described recombinant method for production of each claim as described above, wherein, the fusion product that step a) is produced is incorporated in the expression vector, and this expression vector is applicable to microorganism.

11, recombinant method for production as claimed in claim 10, wherein, described expression vector is applicable to fungi.

12, as claim 10 or 11 described recombinant method for production, wherein, described expression vector comprises inducible promoter or constitutive promoter.

13, as claim 10 or 11 described recombinant method for production, wherein, described expression vector comprises the composing type GAP promotor of pichia pastoris phaff.

14, recombinant method for production as claimed in claim 12, it is characterized in that, the nucleic acid that is obtained is plasmid, and preferred described plasmid is selected from: pPLG11.2, pPLG12.1, pPLG13.1, pPLG14.2, pPLG15.1, pPLG16.3, pPLG17.2, pPLG18.1, pPLG19.2, pPLG20.1, pAC37.1, pJW9.1, pMHS476.1, pSM54.2, pSM49.8, pSM82.1 or pSM58.1.

15, the described recombinant method for production of each claim as described above is characterized in that the restriction enzyme site of described codon proteins encoded enzyme Kex2; The fusion gene of described Profibrinolysin has nucleotide sequence as follows: Seq.ID No.7,13,50,52,54,56 or 58.

16, the described recombinant method for production of each claim as described above is characterized in that the restriction enzyme site of described codon proteins encoded enzyme Kex2; The fusion rotein of described Profibrinolysin has aminoacid sequence as follows: Seq.ID No.8,14,40,42,44,46 or 48.

17, the described recombinant method for production of each claim as described above is characterized in that the restriction enzyme site of described codon proteins encoded enzyme Kex2 and Ste13; The fusion gene of described Profibrinolysin has nucleotide sequence as follows: Seq.ID No.9,15,51,53,55 or 57.

18, the described recombinant method for production of each claim as described above is characterized in that the restriction enzyme site of described codon proteins encoded enzyme Kex2 and Ste13; The fusion rotein of described Profibrinolysin has following aminoacid sequence: Seq.ID No.10,16,41,43,45,47 or 49.

19, the described recombinant method for production of each claim as described above wherein, transforms described microorganism host with the plasmid described in the method for claim 14.

20, the described recombinant method for production of each claim as described above, wherein, employed host is an eukaryotic microorganisms.

21, recombinant method for production as claimed in claim 20 is characterized in that, employed host belongs to fungi.

22, recombinant method for production as claimed in claim 21, it is characterized in that employed host belongs to Pichia (Pichia), yeast belong (Saccharomyces), Hansenula (Hansenula), Candida (Candida) or Aspergillus (Aspergillus).

23, recombinant method for production as claimed in claim 22, it is characterized in that employed host belongs to sow: pichia pastoris phaff (Pichia pastoris), pichia methanolica (Pichia methanolica), multiple-shaped nuohan inferior yeast (Hansenula polymorpha), aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae) or Aspergillus nidulans (Aspergillus nidulans).

24, the described recombinant method for production of each claim as described above, wherein cross encode at least funtion part of Profibrinolysin of the nucleotide sequence of expressing, and the funtion part of described Profibrinolysin at least secreted by a kind of host microorganism, and the fusion product that this host microorganism is produced by step a) transforms and obtains.

25, the described recombinant method for production of each claim as described above, wherein, the funtion part of described Profibrinolysin nucleotide sequence is one of following sequence: Seq.ID No.60,61,62,63,64,65 or 66.

26, the described recombinant method for production of each claim as described above is characterized in that, produces a kind of functional human Profibrinolysin.

27, the described recombinant method for production of each claim as described above is characterized in that, produces a kind of and the functional Profibrinolysin human homology.

28, a kind of plasmid, it is plasmid pPLG11.2, plasmid pPLG12.1, plasmid pPLG13.1, plasmid pPLG14.2, plasmid pPLG15.1, plasmid pPLG16.3, plasmid pPLG17.2, plasmid pPLG18.1, plasmid pPLG19.2 or plasmid pPLG20.1.

29, a kind of plasmid, it is plasmid pMHS476.1, plasmid pSM54.2, plasmid pSM49.8 or plasmid pSM82.1.

30, a kind of plasmid, it is plasmid pSM58.1, plasmid pAC37.1 or plasmid pJW9.1.

31, the nucleotide sequence that obtains according to each described recombinant method for production in the claim 1 to 27 is characterized in that, encode at least the nucleotide sequence of Profibrinolysin funtion part with in yeast, have the coupling of active promotor operability; Also and at least the encode nucleotide sequence coupling of a segment signal peptide of the nucleotide sequence of the described Profibrinolysin funtion part of encoding at least, the nucleotide sequence of described encoding function Profibrinolysin and at least the nucleotide sequence of coded signal peptide by the restriction enzyme site codon coupling of Proteinase K ex2 and Ste13.

32, Profibrinolysin, this Profibrinolysin is obtained by each the described recombinant method for production in the claim 1 to 27.

33, Profibrinolysin as claimed in claim 32 is characterized in that, described Profibrinolysin is Microplasminogen, Miniplasminogen, Methionin Profibrinolysin, L-glutamic acid Profibrinolysin or Profibrinolysin derivative.

34, Profibrinolysin derivative as claimed in claim 33, it is characterized in that, this derivative comprises the functional protein hydrolysis structural domain of Profibrinolysin, and comprise at least one disappearance, and/or at least one amino-acid substitution, and/or with another amino acid or at least one section peptide or at least one protein merge at least.

As claim 33 or 34 described Profibrinolysin derivatives, it is characterized in that 35, described derivative is activated to having active plasmin by at least a plasminogen activator.

36, as each described Profibrinolysin derivative of claim 33 to 35, it is characterized in that, described derivative comprises the functional protein hydrolysis structural domain of Profibrinolysin at least and is higher than 80% with the homology of Microplasminogen, Miniplasminogen, Methionin Profibrinolysin or L-glutamic acid Profibrinolysin, preferably be higher than 90%, especially preferably be higher than 95%.

37, plasmin, this plasmin activates claim each described Profibrinolysin of 32-36 by at least a plasminogen activator or the Profibrinolysin derivative obtains.

38, host microorganism, this host microorganism contain fusion product that each described method of with good grounds claim 1 to 27 obtains or by the arbitrary nucleotide sequence of its deutero-in step a).

39, host microorganism as claimed in claim 38 is characterized in that, this host microorganism is selected from following microorganism: pichia pastoris phaff, pichia methanolica, yeast saccharomyces cerevisiae, multiple-shaped nuohan inferior yeast, aspergillus niger, aspergillus oryzae or Aspergillus nidulans.

40, the functional Profibrinolysin and/or the application of plasmin therefrom in the preparation medicament of producing according to each described method of claim 1 to 27.

41, functional Profibrinolysin of producing according to each described method of claim 1 to 27 and/or plasmin therefrom are used for the treatment of application in the medicine of wound, thrombus incident or pre-preventing thrombosis incident in preparation.

42, functional Profibrinolysin of producing according to each described method of claim 1 to 27 and/or plasmin therefrom are as the application in antithrombotic acitivity agent and the resist coagulation promoting agent.

43, application as claimed in claim 42, this application especially is: prevent and/or treat following disease: heart attack, apoplexy, thrombosis, phlebothrombosis, the heart valve postoperative restenosis, anoxic, ischemic, coagulation necrosis, vascular inflammation, acute pulmonary embolism, acute and subacute arterial thrombus, phlebothrombosis New Development or old is condensed, buttocks and acra deep venous thrombosis, the early stage thrombosis in occluding vascular zone, acute eye central vessel embolism, I type Profibrinolysin lacks the conjunctivitis that causes, burn, alkali or acid burn and frostbite, disseminated intravascular coagulation during shock, acute acra arterial thrombosis, the chronic occlusion arteriopathy, arteriovenous shunt place thrombosis; The successive treatment of following disease: heart attack, bypass surgery, revascularization, balloon dilatation; The thromboembolism treatment of acute cardiac onste; Leading to again of arteriovenous shunt; During the acute cardiac onste, to the coronary artery reperfusion that stops up.

44, as each described application of claim 40 to 43, wherein unite and use described functional Profibrinolysin and/or plasmin therefrom, and antithrombotics.

45, application as claimed in claim 44 is characterized in that, described antithrombotics is heparin, heparin derivatives or acetylsalicylic acid.

46, functional Profibrinolysin of producing according to each described method of claim 1 to 27 and/or plasmin therefrom in joining dressing, paste application or unite application in the use with vulnerary.

47, dressing, wound contain functional Profibrinolysin and/or the plasmin therefrom that each described method of with good grounds claim 1 to 27 is produced with bandage or paste, this dressing, wound with bandage or paste.

48, dressing as claimed in claim 47, wound is with bandage or paste, wherein contains Profibrinolysin and/or the plasmin therefrom of 0.01-500U in every square centimeter of pharmaceutical preparation, is preferably 0.1-250U, is preferably 1-150U especially.

49, claim 47 or 48 described dressing, wound are used for the treatment of application in the medicine of burn, frostbite, alkali or acid burn, damage and/or wound with bandage or paste in preparation.

50, pharmaceutical composition, this pharmaceutical composition contains functional Profibrinolysin and/or the plasmin therefrom that each described method of claim 1 to 27 is produced, with acceptable carrier, additive and/or solvent on the pharmacology, also contain active antithrombotics if desired.

51, pharmaceutical composition as claimed in claim 50, this pharmaceutical composition is applicable to following route of administration: in oral, part or parenteral, the blood vessel, intraperitoneal, subcutaneous or intramuscular, preferred intravenously in the described blood vessel.

52, pharmaceutical composition as claimed in claim 51, when local application, the described pharmaceutical composition of every gram contains 0.01-500U Profibrinolysin and/or plasmin therefrom, is preferably 0.1-250U, is preferably 1-150U especially.

53, pharmaceutical composition as claimed in claim 51, when oral medication, the described pharmaceutical composition of every gram contains 0.1-100, and 000U Profibrinolysin and/or plasmin therefrom are preferably 100-80, and 000U is preferably 1 especially, 000-50,000U.

54, pharmaceutical composition as claimed in claim 51, when injection or transfusion medication, every 10ml solution contains 0.1-100 1,000,000 U Profibrinolysins and/or plasmin therefrom, is preferably 1-10 1,000,000 U, is preferably 3-5 1,000,000 U especially.

55, be used for the treatment of application in the medicine of following disease as each described pharmaceutical composition of claim 50 to 54 in preparation: prevent and/or treat following disease: heart attack, apoplexy, thrombosis, phlebothrombosis, the heart valve postoperative restenosis, anoxic, ischemic, coagulation necrosis, vascular inflammation, acute pulmonary embolism, acute and subacute arterial thrombus, phlebothrombosis New Development or old is condensed, buttocks and acra deep venous thrombosis, the early stage thrombosis in occluding vascular zone, acute eye central vessel embolism, I type Profibrinolysin lacks the conjunctivitis that causes, burn, alkali or acid burn and frostbite, disseminated intravascular coagulation during shock, acute acra arterial thrombosis, the chronic occlusion arteriopathy, arteriovenous shunt place thrombosis; The successive treatment of following disease: heart attack, bypass surgery, revascularization, balloon dilatation; The thromboembolism treatment of acute cardiac onste; In leading to again of arteriovenous shunt; When the acute cardiac onste, to the coronary artery reperfusion that stops up.

56, carrier, this carrier comprise the fusion product that obtained by step a) in the claim 1 or by its nucleotide sequence that derives.

57, dna molecular, this dna molecular comprise the fusion product that obtained by step a) in the claim 1 or by its nucleotide sequence that derives.

58, RNA molecule, this RNA molecule comprise the fusion product that obtained by step a) in the claim 1 or by its nucleotide sequence that derives.

59, identify the screening method of plasminogen activator, this method is used claim 36 or 37 described functional Profibrinolysins.

60, screening method as claimed in claim 59 is characterized in that, after with proteolytic enzyme and claim 35 or 36 described functional Profibrinolysin preincubates, detects the activity of the plasmin that obtains.

61, as claim 59 or 60 described screening methods, it is characterized in that, use synthetic peptide, detect the activity of the plasmin that obtains as substrate.

62, screening method as claimed in claim 61 is characterized in that, uses synthetic peptide N-tosyl-Gly-Pro-Lys-pNA to detect the activity of the plasmin that obtains.