CN101573450B

CN101573450B - The new gene relevant to glutaminyl cyclase

Info

Publication number: CN101573450B
Application number: CN200780034982.4A
Authority: CN
Inventors: S·席林; H·钦尼斯; J-U·拉赫菲尔德; H-U·穆德特
Original assignee: Probiodrug AG
Current assignee: Vivoryon Therapeutics AG
Priority date: 2006-09-21
Filing date: 2007-09-21
Publication date: 2015-12-16
Anticipated expiration: 2027-09-21
Also published as: ZA200901123B; CN101573450A

Abstract

The present invention relates to as glutaminyl cyclase (QC; EC? new glutamine peptide cyclotransferase sample albumen (QPCTL) of isozyme 2.3.2.5); and relate to the isolating nucleic acid of these isozymes of coding; described isolating nucleic acid all for finding new therapeutant, for measuring cyclase activity and inhibit activities for determining the compound for these glutaminyl cyclase isozymes.

Description

The new gene relevant to glutaminyl cyclase

Invention field

The present invention relates to as glutaminyl cyclase (QC; new glutamine peptide cyclotransferase sample albumen (QPCTL) of isozyme EC2.3.2.5); and relate to the isolating nucleic acid of these isozymes of coding; described isolating nucleic acid all can be used for finding new therapeutant, for measuring cyclase activity and for the inhibit activities of deterministic compound to these glutaminyl cyclase isozymes.

Background of invention

Glutaminyl cyclase (QC, EC2.3.2.5) catalyze ammonia cardinal extremity glutamine residue is to Pyrrolidonecarboxylic acid (pGlu ^*) intramolecular cyclization, thus release ammonia.QC was separated (Messer, M.1963Nature4874,1299) by Messer first the emulsion from tropical plants papaya (Caricapapaya) in 1963.After 24 years, corresponding enzymic activity finds in animal hypophysis (1987JBiolChem262, the 8532-8536 such as Busby, W.H.J.; Fischer, W.H. and Spiess, J.1987ProcNatlAcadSciUSA84,3628-3632).For Mammals QC, can confirm that QC causes Gln to be converted into pGlu (1987JBiolChem262, the 8532-8536 such as Busby, W.H.J. at the precursor of TRH and GnRH; Fischer, W.H. and Spiess, J.1987ProcNatlAcadSciUSA84,3628-3632).In addition, initial QC positioning experiment shows it and locates altogether with the presumption product of its katalysis in Niu Chuiti, this promotes further its potential function in peptide hormone synthesis 1995JNeuroendocrinol7,445-453 such as () Bockers, T.M..On the contrary, the physiological function of plant QC is more unclear.With regard to the enzyme from papaya (C.papaya), effect 2001CellMolLifeSci58,556-570 such as () ElMoussaoui, A. in the plant defense of opposing pathogenic microbes is proposed.The presumption QC (2000ProteinExprPurif20, the 27-36 such as Dahl, S.W.) from other plant is identified more recently by gene comparision.But the physiological function of these enzymes is still unclear.

From the known QC display of plant and animal, the stringent specificity of L-glutaminate in substrate N-terminus position is found that their dynamics observes Michaelis-Menten equation (Pohl, T. 1991ProcNatlAcadSciUSA88,10059-10063 is waited; The 1988AnalBiochem175 such as Consalvo, A.P., 131-138; 1996BiolChemHoppeSeyler377, the 395-398 such as Gololobov, M.Y.).But, compare primary structure from the QC of papaya and the primary structure from mammiferous well-conserved QC and then do not disclose any sequence homology 2000ProteinExprPurif20,27-36 such as () Dahl, S.W..Although plant QC seems to belong to a new family (Dahl, S.W. 2000ProteinExprPurif20 is waited, 27-36), but find that Mammals QC has the remarkable sequence homology (Bateman with bacterium aminopeptidase, R.C. 2001Biochemistry40 is waited, 11246-11250), thus draw conclusion like this, the QC namely from plant and animal has Different Evolutionary origin.

Recently, recombinant human QC has been confirmed and from the QC-active catalytic aminoterminal glutamyl of brain extract and L-glutamic acid cyclisation.The most surprisingly find near pH6.0, be conducive to the enzymatic Glu of cyclisation ₁-transform, and Gln ₁conversion to pGlu derivative occurs optimal pH about 8.0 time.Due to can by suppress recombinant human QC active and from the QC of porcine pituitary extract activity and suppress pGlu-A beta related peptides to be formed, therefore QC enzyme develops the target being used for the treatment of the medicine of alzheimer's disease.

EP02011349.4 discloses the polynucleotide of coding insect glutaminyl cyclase and coded polypeptide thereof.Also providing package is containing the host cell of expression vector for this application, and wherein said expression vector comprises polynucleotide of the present invention.Isolated polypeptide and comprise insect QC host cell for screening in the method for the material reducing glutaminyl cyclase activity.This type of material is used as sterilant.

The QC inhibitor that also can be used as the inhibitor of QC isozyme describes in WO2004/098625, WO2004/098591, WO2005/039548 and WO2005/075436, above-mentioned document is complete to be incorporated herein, especially like this in the structure of described inhibitor, purposes and generation thereof.

Definition

Enzyme inhibitors

Reversibility enzyme inhibitors: comprise competitive inhibitor, reversible non-competitive inhibition agent slow fixation or close adhesion inhibitor, transition state analog and multiple substrate analogue.

competitive inhibitordisplay

I) interact with enzyme non-covalent,

Ii) with substrate competition zymophore,

The Main Function mechanism of reversibility enzyme inhibitors and the definition of dissociation constant can be shown as follows:

The formation of enzyme-inhibitor [E-I] mixture stops Binding Capacity, and thus this reaction can not be advanced into normal physiological product P.Larger inhibitor concentration [I] produces comparatively large [E-I], thus make can be less with the resolvase of Binding Capacity.

reversible non-competitive inhibition agent

I) (allosteric combining site) is combined at other position except reactive site

Ii) the enzyme conformational change reducing or stop catalytic activity is caused

slow fixation or close adhesion inhibitor

I) be the competitive inhibitor that the balance wherein between inhibitor and enzyme slowly reaches,

Ii) (k _onslowly), may owing to the conformational change that must occur in enzyme or inhibitor

A) transition state analog often

B) with enzyme concn (sub-nmole K _dvalue) in similar concentration effectively

C) because of k _offbe worth so low, therefore the inhibitor of these types " almost " is irreversible.

transition state analog

It is the competitive inhibitor of the transition state of mimetic enzyme catalysis reaction.Enzyme catalysis occurs because reducing the energy of this transition state, and therefore, transition state keying action has precedence over Binding Capacity effect.

multiple substrate analogue

For the reaction relating to two or more substrates, can design such competitive inhibitor or transition state analog, it contains the constitutional features similar to two or more substrates described.

Irreversible enzyme inhibitor: always drive the balance between unconjugated enzyme and inhibitor and enzyme inhibitor complex (E+I<--->E-I) to the right, containing making the irreversible covalent linkage of restraining effect (about 100kcal/ mole).

affinity tag

. for the irreversible inhibitor of reactive site(competitive irreversible inhibitor), by enzyme identification (reversibility, specific binding), forms covalent linkage subsequently, and

I) structurally similar to substrate, transition state or product, thus create conditions for specificity between medicine and target enzyme interacts,

Ii) reactive functional groups (such as the nucleophilic group ,-COCH that create conditions is formed containing promising covalent linkage ₂br).

Following reaction scheme describes reagent for reactive site and its target enzyme, wherein K _dbe dissociation constant and k _deactivation(k _inactivation) be covalent linkage synthesis speed.

. based on the enzyme-deactivating thing (being also called suicide inhibitor) of mechanismbe the reagent (non-reacted) for reactive site, this reagent and wherein this reagent change into activity form (activation) zymophore because of enzyme catalysis ability is combined.Once activate, then form covalent linkage between enzyme therewith at described inhibitor.

Following reaction scheme shows the mechanism of action based on the enzyme-deactivating thing of mechanism, wherein K _dthe mixture that dissociates, k ₂be with enzyme in conjunction with time activation inhibitor speed, k ₃be the inhibitor P the activated speed (product still can responding property) of dissociating from this enzyme and k ₄the speed forming covalent linkage between inhibitor and enzyme activated.

(covalent linkage is formed, k in deactivation ₄) must at (the k that dissociates ₃) occur before, otherwise activity inhibitor is released in environment now.For side reaction more than effective inactivation system and undesirable Minimum Residual, partition rate k ₃/ k ₄, namely releasing product is to deactivation product ratio, should minimize.Large partition rate (support is dissociated) causes nonspecific reaction.

Noncompetitive enzyme inhibitors: following equation can be write out from the definition of noncompetitive inhibitor (inhibitor be only combined with ES mixture):

Described ES mixture to dissociate described substrate with the dissociation constant equaling Ks, this substrate (namely having null Ks value) and ESI mixture does not dissociate.The K of expection Michaelis-Menten type enzyme _mreduce.Increasing concentration of substrate causes ESI (can not develop into the mixture of reaction product) concentration to increase, therefore can not derepression effect.

According to the present invention, preferred competitiveness enzyme inhibitor.

Most preferably competitive reversibility enzyme inhibitors.

Term " k _i" or " K _i" and " K _d" be describe inhibitor to be combined with enzyme and the binding constant discharged from enzyme subsequently.Another kind of tolerance is " IC ₅₀" value, this value is reflected in the inhibitor concentration given concentration of substrate producing 50% enzymic activity.

As used herein, term " QC " comprises the glutaminyl cyclase (QC) with glutamine peptide cyclotransferase (QPCT) synonym; And with the QC sample enzyme of glutamine peptide cyclotransferase sample albumen (QPCTL) synonym.QC and QC sample enzyme has same or analogous enzymic activity, and this enzymic activity is defined as again QC activity.With regard in this respect, QC sample enzyme can be basic different from QC on its molecular structure.

" QC-is active " is defined as the catalytic activity of glutaminyl cyclase (QC, QPCT) and QC sample enzyme (QPCTL).In the Various Tissues (comprising kidney, liver, intestines, brain) that these enzymes are present in body of mammals and body fluid (as CSF), wherein said enzyme is in glutamine or the L-glutamic acid at the aminoterminal place of biologic activity peptide with high degree of specificity catalysis.

Especially, " QC active " to be defined as when discharging ammonia aminoterminal glutamine residue to Pyrrolidonecarboxylic acid (pGlu as used herein, the term ^*) intramolecular cyclization or aminoterminal L-height glutamine or L-β-Gao glutamine to the intramolecular cyclization of cyclic diolefin height Glutamine Derivatives.Thus see scheme 1 and 2.

Scheme 1:QC is to the cyclisation of glutamine

Scheme 2:QC is to the cyclisation of L-height glutamine

" EC " comprises glutaminyl cyclase (QC as used herein, the term; QPCT) and QC sample enzyme (QPCTL) as the pair activity (sideactivity) of glutamate cyclase (EC), this activity is defined as again EC activity.

" EC active " is defined as glutaminyl cyclase (QC, QPCT) and QC sample enzyme (QPCTL) makes aminoterminal glutaminic acid residue generation intramolecular cyclization become Pyrrolidonecarboxylic acid (pGlu as used herein, the term ^*).Therefore see scheme 3.

Scheme 3:QC (EC) is to the aminoterminal cyclisation of uncharged glutamyl peptide

Term " QC inhibitor " or " glutaminyl cyclase inhibitor " are that those skilled in the art are usually known and mean such enzyme inhibitors; it suppresses glutaminyl cyclase (QC; QPCT) or the catalytic activity of QC sample enzyme (QPCTL) or its glutamyl cyclase (EC) active, carry out preferably by this inhibitor and described enzyme direct interaction.

Term " selectivity QC inhibitor " means such enzyme inhibitors as defined herein; it suppresses glutaminyl cyclase (QC; QPCT) catalytic activity, but do not suppress or suppress at least one QC sample enzyme (QPCTL) with lower ability.Preferably such selectivity QC inhibitor, this inhibitor suppresses glutaminyl cyclase (QC, QPCT) to suppress the ki value of the low order of magnitude of the ki value of at least one QC sample enzyme (QPCTL) than it.More preferably, described selectivity QC inhibitor suppresses the ki-value of glutaminyl cyclase (QC, QPCT) two orders of magnitude lower than the ki value of its suppression at least one QC sample enzyme (QPCTL).Even more preferably such selectivity QC inhibitor, wherein they suppress the ki-value of glutaminyl cyclase (QC, QPCT) three orders of magnitude lower than the ki value of its suppression at least one QC sample enzyme (QPCTL).Most preferably do not suppress the selectivity QC inhibitor of QC sample enzyme (QPCTL).

Term " selectivity QPCTL-inhibitor " means such enzyme inhibitors as defined herein; it suppresses the catalytic activity of at least one QC sample enzyme (QPCTL); but do not suppress or suppress with lower ability the activity of glutaminyl cyclase (QC, QPCT).Preferably such selectivity QPCTL-inhibitor, this inhibitor suppresses the ki value of at least one QC sample enzyme (QPCTL) order of magnitude lower than the ki value of its suppression glutaminyl cyclase (QC, QPCT).More preferably, described selectivity QPCTL-inhibitor suppresses the ki-value of at least one QC sample enzyme (QPCTL) two orders of magnitude lower than the ki value of its suppression glutaminyl cyclase (QC, QPCT).Even more preferably such selectivity QPCTL-inhibitor, wherein they suppress the ki-value of at least one QC sample enzyme (QPCTL) three orders of magnitude lower than the ki value of its suppression glutaminyl cyclase (QC, QPCT).Most preferably do not suppress the selectivity QC inhibitor that glutaminyl cyclase (QC, QPCT) is active.

The ability that QC suppresses

Just with QC restraining effect relatedly, in preferred embodiments, present method or medical usage utilize such material, and it has 10 μMs or less, more preferably 1 μM or less, even more preferably 0.1 μM or less or 0.01 μM or less or the most preferably QC restraining effect K of 0.01 μM or less _i.In fact, contemplate and have at less micromolar levels, K preferably at nanomolar range and even more preferably within the scope of picomolar _ithe inhibitor of value.Therefore, although for convenient object, active substance is described as in this article " QC inhibitor ", but is to be understood that this type of name is not intended to theme of the present invention to be limited to the concrete mechanism of action.

The molecular weight of QC inhibitor

Typically, the QC inhibitor of present method or medical usage can be small molecules, such as, there is 1000g/ mole or less, 500g/ mole or less, preferred 400g/ mole or less and even more preferably 350g/ mole or less and even 300g/ mole or less molecular weight.

" object " refers to that preferred mammal, optimum is chosen as the animal for the treatment of, observation or experiment object as used herein, the term.

As used herein, the term " treatment significant quantity " mean to probe into researcher, animal doctor, doctor or clinician tissue system, excite the active compound of biological response or medical science response or the amount of medicinal substance in animal or human, described biological response or medical science response comprise the symptom of disease that improvement treating or illness.

As used herein, term " pharmaceutically useful " comprises people's purposes and veterinary purpose beast: such as term " pharmaceutically useful " comprises the acceptable compound of animal doctor or acceptable compound in people's medical science and health care.

Guillain-Barr é syndrome (GBS)

Alternative names is Landry-Guillain-Barr é syndrome, acute idiopathic polyneuritis, infectious polyneuritis or acute inflammation polyneuropathy.

Guillain-Barr é syndrome is a kind of serious conditions occurred when neural part is attacked in body defenses (immunity) system mistake.This causes and causes amyasthenic neuroinflamation, and wherein the latter continues to worsen.

Guillain-Barr é syndrome is autoimmune disorder.The syndromic clear and definite cause of disease of unknown Guillain-Barr é.This syndrome can occur at any age, but 30 and two kinds of sexes of 50 years old crowd in the most common.It is secondary mild infection usually, and normally respiratory tract (lung) infects or digestive tube (gi tract are long) infection.Usually, the initial sign that infects disappears before the symptom of Guillain-Barr é starts.Guillain-Barr é syndrome causes the inflammation of injured nerve part.This nervous lesion causes tingle, myasthenia and paralysis.The tectum (myelin) that this inflammation affects the nerves usually.This infringement is called demyelination.Demyelination slows down Neurotransmission effect.Nerve can be caused to quit work to the infringement of neural other parts.

The symptom of Guillain-Barr é worsens extremely rapidly.Namely it only can reach the most serious symptom in several hours.Myasthenia or muscle function are lost (paralysis) and are affected health both sides.If myasthenia starts at leg and extends to arm subsequently, then this myasthenia is called ascending paralysis.

Patient can aware tingle, podalgia or hand pain and clumsiness.When muscle function forfeiture increases the weight of, this patient may need assisted respiartion.

Guillain-Barr é syndrome cannot be cured.But numerous therapy can be used for helping mitigation symptoms, and complication also adds quick-recovery.When serious symptom, needs of patients is in hospital with assisted respiartion, treatment and physiotherapy.Use the method being called plasmaphoresis to take out blood samples of patients and to replace not containing the intravenous fluid of antibody or the blood of donations.The immunoglobulin therapy of high dosage is used to reduce the seriousness of Guillain-Barr é symptom and the another kind of method of time duration.Other therapies is intended to complication prevention.

Chronic inflammatory demyelinating polyneuropathy (CIDP)

A kind of similar to GBS but be that the disease of feature is called chronic inflammatory demyelinating polyneuropathy (CIDP) with chronic process.For CIDP, the also general definition be suitable for, but observe that it is contrary with GBS, its progressive stage, is continued above 4 weeks, often beyond 6 months, and often leaves defect in patients.Because GBS and CIDP causes serious paretic mechanism to comprise to be secondary to the immune response of T mediated by lymphocytes and the inflammation of peripheral nerve unit demyelinization.This hypothesis by the amount of the complement complex observed in patients serum and cerebrospinal fluid or cytokine raise support.At present by demyelination process, the demyelination process especially in nerve root region is considered as the developing decisive mechanism of nerve block.Based on a kind of theoretical step relatively important in early days in development using blood/cerebrospinal fluid (CSF) barrier as this disease.Another kind of theory claims that the leakage at blood/CSF barrier develops because of this disease and causes protein content increase in CSF.In a word, not being directly involved in immune nonspecific sera component can infiltrate CSF from blood, causes neurone or spongiocyte functional disorder and/or regulates neuronal activity.Another kind of mechanism is that CSF flow velocity reduces, and the protein content in this possible explanation CSF increases.This explanation does not require that blood/CSF barrier selectivity is impaired or change.Although mentioned whole effects may be important for GBS and CIDP process, but they are not still illustrated the actual contribution of symptom.Also can not set up contacting between the protein concn increased in CSF and certain electric physiological Study result or clinical picture.Be described in recently in the CSF of GBS patient and patients with multiple sclerosis with the interactional factor in action potential dependency sodium channel (W ü z etc. 1995, MuscleandNerve18,772-781).The factor described in Brinkmeier (Brinkmeier etc. 1996, MuscleandNerve19,54-62) report has and is less than 3kDa molecular weight, and is less than 1kDa under stricter experiment condition.Even if observe and the fact of described factor active also not obvious reduction after CSF and protease incubation based on this, author reaches a conclusion; The described factor is non-antibody also non-cytokine both.

Multiple sclerosis (MS)

Multiple sclerosis is the autoimmune disorder affecting central nervous system (brain and spinal cord).The impact of multiple sclerosis on women exceedes the male sex usually.This illness is the most common to start between 20 years old-40 years old, but can show effect on any age.The definite cause of disease is unknown, but it is believed that MS is caused by the protectiveness material-myelin infringement because surrounding neurocyte.This disease is PD, means that this infringement increases the weight of in time.Inflammation destroys myelin, leaves multiple scar tissue district (sclerosis).When the immune cells attack neural system of health self, inflammation occurs.This inflammation causes the slack-off or retardance of nerve impulse, produces MS symptom.The recurrent exerbation of inflammation or burst can occur along region any in brain and spinal cord.Symptom is changeable, because the position of each invasion and attack is different with degree.Usually, last for days, several weeks or several months outbreak and symptom occurrence number declines or asymptomatic (alleviation) alternately occurs.(recurrence) is common repeatedly, but also can occur without the continuing advances alleviating period.

Which kind of material not clear starts is attacked.MS patient generally has higher immuning cell number than Healthy People, and this prompting immunne response may play a role.Modal theory relates to virus or hereditary defect or its and combines.This disease seems also there is genetic linkage.Compared with other area, MS more may appear at the north, Europe, northern US, South Australia and New Zealand.Geography research shows there is relevant environmental factor.There is the people of MS family history and MS sickness rate those people that live in district higher, there is this higher sick risk.

Knownly at present multiple sclerosis cannot be cured.But, there are numerous therapies of this disease that can slow down.Therapeutic purpose control symptom and maintain orthobiosis quality.

Invention summary

The invention provides the protein with glutaminyl cyclase activity; the newcomer of the protein families that its composition is relevant to glutaminyl cyclase, described protein comprises full-length proteins, alternative splice forms, subunit and mutant and their nucleotide sequence of encoding.The present invention also provides the method for screening substrate, interaction protein, the agonist of above-mentioned protein, antagonist or inhibitor, and relates to the pharmaceutical composition comprising described protein and/or mutant, its derivative and/or analogue and/or its part.

With the glutaminyl cyclase (nucleotide sequence of SEQIDNO:1, the protein sequence of SEQIDNO:10) these new proteins with remarkable sequence similarity are protein (QPCTL) (also known as making people isoQC) (GenBank accession number NM_017659) from people, from the protein (GenBank accession number NM_027455) of mouse, from the protein (GenBank accession number AB168255) of cynomolgus monkey (Macacafascicularis), from the protein (GenBank accession number XM_001110995) of macaque (Macacamulatta), from the protein (GenBank accession number XM_541552) of cat, from the protein (GenBank accession number XM_001066591) of rat, from the protein (GenBank accession number BT026254) of milk cow or its, there is at least 50%/75% sequence thereto/similarity, preferably 70%/85% sequence thereto/similarity, the analogue of most preferably 90%/95% sequence thereto/similarity.

Protein sequence provides in SEQIDNO:11-18.Further disclose these nucleic acid sequences to proteins of coding (SEQIDNO:2-9).Table 1 shows the similarity between described new protein and known glutaminyl cyclase.The aobvious homogeny stated between described new protein and known glutaminyl cyclase of table 2.

Table 1: the similarity of new glutamine peptide cyclotransferase sample albumen and the protein sequence of glutaminyl cyclase

QPCTL originates	People isoQC (SEQ ID NO:11)	People QC (SEQ ID NO:10)
			People isoQC (SEQ ID NO:11)	-	71.98％
Cynomolgus monkey (SEQ ID NO:13)	99.48％	72.24％
			Macaque (SEQ ID NO:14)	99.48％	72.24％
Domesticated dog (C_familiaris) (SEQ ID NO:15)	95.82％	72.31％
			Rattus norvegicus (R_norvegicus) (SEQ ID NO:16)	95.30％	70.77％
Mouse (M_musculus) (SEQ ID NO:17)	95.04％	70.77％
			Family ox (B_taurus) (SEQ ID NO:18)	96.08％	72.31％

Table 2: the homogeny of new glutamine peptide cyclotransferase sample albumen and the protein sequence of glutaminyl cyclase

QPCTL originates	People isoQC (SEQ ID NO:11)	People QC (SEQ ID NO:10)
			People isoQC (SEQ ID NO:11)	-	45.24％
Cynomolgus monkey (SEQ ID NO:13)	98.17％	44.99％
			Macaque (SEQ ID NO:14)	98.17％	44.99％
Domesticated dog (C_familiaris) (SEQ ID NO:15)	88.51％	45.13％
			Rattus norvegicus (R_norvegicus) (SEQ ID NO:16)	84.33％	45.38％
Mouse (M_musculus) (SEQ ID NO:17)	84.07％	44.62％
			Family ox (B_taurus) (SEQ ID NO:18)	84.60％	45.64％

The high similarity of 95-99% and the perfect homology (see Fig. 2) of 84-98% is there is between the QPCTL of different sources.Based on the sequence similarity (see Fig. 1) of same human glutaminyl cyclase and mouse glutaminyl cyclase, can predict that the function that these QPCTL may have includes, but are not limited to, as enzyme.Clone, expression, Biochemical Characterization and the verified this hypothesis of characterization of molecules.

The expression pattern of QPCTL in brain, prostate gland and lung tissue is consistent with the function in hereinafter described disease.Enzymic activity as glutaminyl cyclase shows QPCTL activity or inhibition molecule has multiple therepic use, as mentioned below.

To play function affect as the enzyme modification effect of hormone, peptide and chemokine as the physiological regulator of immunity system and neuroendocrine system by biochemical mediators mutually compatible with them for the active and expression pattern of QPCTL described herein.The numerous functions previously based on using inhibitor, QC described can partly owing to the effect of QC and similar protein matter as the effect of QPCTL.Therefore, think that the selectivity that finds QC, QPCTL or other relevant enzyme and potent inhibitor realize the glutamine peptide cyclotransferase of these enzymes and any new qualification and the effective of other active compound and the key of safe pharmaceutical usage, other wherein said active compound modifies the function of this proteinoid.

Therefore the present invention provides new protein or polypeptide, encode they nucleic acid, with described nucleic acid modified to express these protein cell, for the antibody of these protein, for finding the screening method of new therapeutant, the therapeutant that wherein said new therapeutant is the inhibitor of the activity of these protein (or they are the inhibitor of QC instead of the inhibitor of described protein) and is found by this type of screening side.Described new protein and their nucleic acid of coding can be used for finding the new therapeutant of some disease for the treatment of (such as neurodegenerative disease, reproduction disease, inflammatory diseases and metabolic disease) and being used in the antibody that preparation has therapeutic value or diagnostic value.

According to an aspect of the present invention, new, ripe biological activity protein is provided, preferred human origin.This proteinoid marginally can be separated from suitable animal (comprising people) tissue or biofluid by standard technique; But, larger amount more easily through genetically engineered with the culture of the cell of expressing described protein in prepare.

According to another aspect of the present invention, provide the isolated nucleic acid molecule of code book invention polypeptide, described isolated nucleic acid molecule comprises its mRNA, DNA, cDNA, genomic dna.

According to a further aspect of the invention, also provide nucleic acid probe, it comprises the enough nucleic acid molecule with nucleotide sequence specific hybrid of the present invention of length.

According to a further aspect of the invention, provide the method for use recombinant technology to produce this type of polypeptide for external scientific research (such as synthetic DNA and manufacture DNA vector).The restructuring protokaryon through DNA vector transfection and/or eukaryotic host cell is cultivated be included in the condition promoting to express described protein for generation of the method for this type of polypeptide under, wherein said DNA vector contains the nucleotide sequence of this peptide species of coding and/or maturation protein, and reclaims the fragment of this protein or expression product subsequently.

According to another aspect, the invention provides the method using QPCTL polypeptide and polynucleotide therapy disease.

According to a further aspect of the invention, the method utilizing the polynucleotide of this type of polypeptide or encoding such polypeptides to find compound like this is provided, wherein said compound suppresses the biologic activity of described maturation protein, and such as QC is active or EC is active, and therefore also provides this type of inhibitor.

According to aspect more specifically, the invention provides the nucleic acid of separation, this nucleic acid encoding (a) is selected from the QPCTL polypeptide of SEQIDNO:11-18, or coding (b) polypeptide, it has and has the aminoacid sequence at least about 75% similarity with the aminoacid sequence of described QPCTL polypeptide and show identical biological function, or this nucleic acid is the alternative splice variants of one of SEQIDNO:2-9; Or this nucleic acid is the probe of at least 14 continuous nucleotides of the described nucleic acid comprising to come own coding (a) or (b); Or the complementary nucleic acid of this nucleic acid and aforementioned any one.

According to another concrete aspect, the invention provides such polypeptide, it can be optionally glycosylated, and its (a) has the aminoacid sequence of the arbitrary shown maturation protein of SEQIDNO:10-18, the preferably arbitrary shown maturation protein of SEQIDNO:11-18; B () has the aminoacid sequence having at least about 75% similarity with one of the maturation protein of (a) and show the maturation protein of identical biological function; C () has the aminoacid sequence of maturation protein like this, wherein said ripe protein and the arbitrary maturation protein of SEQIDNO:10-18, preferably SEQIDNO:11-18 arbitrary shown in maturation protein have at least about 50% homogeny; Or (d) its be the immunological response fragment of (a)

According to another concrete aspect, the invention provides the method that screening can suppress the compound of the enzymic activity of at least one maturation protein of the present invention, described maturation protein is preferably selected from the protein of SEQIDNO:11-18, described method is included in the suitable substrates of maturation protein and this maturation protein described in incubation when there is test compounds or its salt described in one or more, measure the enzymic activity of this maturation protein, this is active in determined commeasurable expression activitiy when there is not test compounds, and select one or more test compounds reducing described enzymic activity.

In addition, the present invention relates to the diagnostic kit based on the purposes of QC inhibitor, selectivity QC inhibitor or selectivity QPCTL inhibitor and method.

Those skilled in the art from the following detailed description should obviously these aspects of the present invention and other side.

Accompanying drawing is sketched

Fig. 1 shows people QC (hQC), the sequence alignment result of people isoQC (hisoQC), mouse QC (mQC) and mouse isoQC (misoQC).Be used in PBIL ( bioinformatiqueLyonnais) ClustalW of (http://npsa-pbil.ibcp.fr) carries out Multiple sequence alignments with default setting.The conservative property of the residue of connection zine ion is with runic and underline mode to people QC (hQC; GenBankX71125, SEQIDNO:10), people isoQC (hisoQC, GenBankNM_017659, SEQIDNO:11), mouse QC (mQC, GenBankNM_027455, SEQIDNO:79) and mouse isoQC (misoQC, GenBankBC058181, SEQIDNO:17) display.

Fig. 2 shows the sequence alignment result of following isoQC: from the isoQC (hisoQC of homo sapiens (Homosapiens), GenBankNM_017659, SEQIDNO:11), from the isoQC (M_fascicularis of cynomolgus monkey, GenBankAB168255, SEQIDNO:13), from the isoQC (M_mulatta of macaque, GenBankXM_001110995, SEQIDNO:14), from the isoQC (C_familiaris of domesticated dog (Canisfamiliaris), GenBankXM_541552, SEQIDNO:15), from the isoQC (R_norvegicus of Rattus norvegicus (Rattusnorvegicus), GenBankXM_001066591, SEQIDNO:16), from the isoQC (M_musculus of mouse (Musmusculus), GenBankBC058181, and the isoQC (B_taurus of family ox (Bostaurus) SEQIDNO:17), GenBankBT026254, SEQIDNO:18).Be used in PBIL ( bioinformatiqueLyonnais) ClustalW of (http://npsa-pbil.ibcp.fr) carries out Multiple sequence alignments with default setting.Residue amino acid conservative property being connected to zine ion underlines and bold print.

Fig. 3 shows the sequence alignment result of other M28 family member of people QC (hQC, SEQIDNO:10) and people isoQC (hisoQC, SEQIDNO:12) and metallopeptidase ClanMH.The ClustalW being used in ch.EMBnet.org carries out Multiple sequence alignments with default setting.People QC (hQC; Swiss-ProtQ16769, SEQIDNO:10) in connect the conservative property of amino-acid residue of single zine ion to people isoQC (isoQC; Swiss-ProtQ53HE4, SEQIDNO:12) (19-382 position residue), Zn dependency aminopeptidase (SGAP from streptomyces griseus (Streptomycesgriseus); Swiss-ProtP80561, SEQIDNO:80) and the ripe Zn-dependency leucinyl amino group peptase (VpAP of vibrio proteolyticus (Vibrioproteolyticus); Swiss-ProtQ01693, SEQIDNO:81) display.Corresponding amino-acid residue is underlined and bold print.

Fig. 4 shows the sequence alignment result of people QC (hQC, SEQIDNO:10) and people isoQC (hisoQC, SEQIDNO:11), shows two presumption property translation starting points and (methionine(Met) I-runic, not to underline; Methionine(Met) II-runic).Be used in PBIL ( bioinformatiqueLyonnais) http:// npsa-pbil.ibcp.frclustalW carry out Multiple sequence alignments with default setting.The membrane spaning domain existed in people isoQC is marked by black rod.

Fig. 5 shows the sequence alignment result of people QC (hQC, SEQIDNO:10) and people isoQC (hisoQC, SEQIDNO:12), from methionine(Met) II (runic).The ClustalW being used in ch.EMBnet.org carries out multiple sequence with default setting.The amino acid participating in melts combine underlines and bold print.The membrane spaning domain existed in people isoQC is marked by black rod.

Fig. 6 shows the analysis expressed isoQC by RT-PCR.Detect in SH-SY5Y, LN405, HaCaT and Hep-G2.

Swimming lane: bp, DNA standard; 1, from the pcr amplification product of the people isoQC of SH-SY5Y; 2, from the pcr amplification product of the people isoQC of LN405; 3, from the pcr amplification product of the people isoQC of HaCaT; 4, from the pcr amplification product of the people isoQC of Hep-G2.

Fig. 7 display is by the analysis of immunohistochemistry to isoQC (MetI, SEQIDNO:11) Subcellular Localization.The people isoQC (see Fig. 5) started at methionine(Met) I place is expressed as the fusion rotein (isoQC (MetI) EGFP) of band EGFP in LN405.AB3712 (Chemicon) is used to carry out mannosidase II redyeing process.Merge the overlap representing isoQC (MetI)-EGFP and mannosidase II staining.

Fig. 8 display is by the analysis of immunohistochemistry to isoQC (MetI, SEQIDNO:11) Subcellular Localization.The people isoQC started at methionine(Met) I place is expressed as the fusion rotein (isoQC (MetI) EGFP) of band EGFP in LN405.MAB1273 (Chemicon) is used to carry out plastosome redyeing process.Merge the overlap representing isoQC (MetI)-EGFP and plastosome staining.

Fig. 9 display is by the analysis of immunohistochemistry to isoQC (MetII, SEQIDNO:12) Subcellular Localization.The people isoQC started at methionine(Met) II place is expressed as the fusion rotein (isoQC (MetII) EGFP) of band EGFP in LN405.AB3712 (Chemicon) is used to carry out dew Glycosylase II redyeing process.Merge the overlap representing isoQC (MetII)-EGFP and mannosidase II staining.

Figure 10 display is by the analysis of immunohistochemistry to isoQC (MetII, SEQIDNO:12) Subcellular Localization.The people isoQC started at methionine(Met) II place is expressed as the fusion rotein (isoQC (MetII) EGFP) of band EGFP in LN405.MAB1273 (Chemicon) is used to carry out plastosome redyeing process.Merge the overlap representing isoQC (MetII)-EGFP and plastosome staining.

Figure 11 display is by the analysis of immunohistochemistry to isoQC (MetI, SEQIDNO:11) Subcellular Localization.The people isoQC started at methionine(Met) I place is expressed as the fusion rotein (isoQC (MetI) EGFP) of band EGFP in COS-7.AB3712 (Chemicon) is used to carry out mannosidase II redyeing process.Merge the overlap representing isoQC (MetI)-EGFP and mannosidase II staining.

Figure 12 display is by the analysis of immunohistochemistry to isoQC (MetI, SEQIDNO:11) Subcellular Localization.The people isoQC started at methionine(Met) I place is expressed as the fusion rotein (isoQC (MetI) EGFP) of band EGFP in COS-7.MAB1273 (Chemicon) is used to carry out plastosome redyeing process.Merge the overlap representing isoQC (MetI)-EGFP and plastosome staining.

Figure 13 display is by the analysis of immunohistochemistry to isoQC (MetII, SEQIDNO:12) Subcellular Localization.The people isoQC started at methionine(Met) II place is expressed as the fusion rotein (isoQC (MetII) EGFP) of band EGFP in COS-7.AB3712 (Chemicon) is used to carry out mannosidase II redyeing process.Merge the overlap representing isoQC (MetII)-EGFP and mannosidase II staining

Figure 14 display is by the analysis of immunohistochemistry to isoQC (MetII, SEQIDNO:12) Subcellular Localization.The people isoQC started at methionine(Met) II place is expressed as the fusion rotein (isoQC (MetII) EGFP) of band EGFP in COS-7.MAB1273 (Chemicon) is used to carry out plastosome redyeing process.Merge the overlap representing isoQC (MetII)-EGFP and plastosome staining.

Figure 15 shows the conversion that inhibitor P150/03 suppresses the H-Gln-AMC to pGlu-AMC of people isoQC catalysis.Assuming that linear competitive inhibition, according to Michaelis-Menten dynamicmodel evaluating data.Inhibitor concentration is as follows:

The K measured _i-value is 240 ± 8nM.

Figure 16 shows the conversion of the people isoQC-catalysis H-Gln-Ala-OH to pGlu-Ala-OH using spectrophotometer measurement assay method to measure.According to Michaelis-Menten dynamicmodel evaluating data.Kinetic parameter is for K _mand V _max-value is 324 ± 28 μMs and 7.4 ± 0.2nM/ minute respectively.

Figure 17 is provided in the scheme of the people isoQC protein construct of heterogenous expression in pichia pastoris phaff (P.pastoris).In some protein, import two sudden changes, produce glycosylation site at the 55th position (I55N) place and produce the cysteine residues suddenlyd change at the 351st position (C351A) place.In order to express, replace with the secretion signal of yeast (YSS) aminoterminal comprising membrane spaning domain.Construct containing this aminoterminal secretion signal should be secreted effectively in substratum.

It is active that Figure 18 is presented at the QC occurring to measure in the substratum of the yeast cell of expressing.Natural construct is not secreted because of membrane spaning domain to substratum (enforcement).Protein because of glycosylation (I55N), secrete efficiently by pole.Sudden change C351A also causes detecting that higher QC is active in the medium.Described construct describes in fig. 17.

Figure 19 display is based on construct YSShisoQCI55NC351AC-His, and people isoQC is purifying from the substratum of transgenosis pichia pastoris phaff strain.This QC carries out purifying by the combination of IMAC (immobilized metal affinity chromatography, swimming lane 3), HIC (hydrophobic interaction chromatography, swimming lane 4) and desalination (swimming lane 5).The glycosylation of this enzyme is confirmed by enzyme deglycosylation, and wherein said enzyme deglycosylation causes the movement of protein migration (swimming lane 6).Swimming lane 1, protein standards: swimming lane 2, the substratum before purifying.

Figure 20. show based on building GST-hisoQCC-His, people isoQC purifying from the cell homogenates of the intestinal bacteria (E.coli) transformed.This QC by IMAC (immobilized metal affinity chromatography, swimming lane 3), GS is affine (swimming lane 4), purifying is carried out in the combination of desalination (swimming lane 5) and ion exchange chromatography (swimming lane 6).Swimming lane 1, protein standards: swimming lane 2, the cell homogenates before purifying.In yeast and intestinal bacteria, between expressed hisoQC, the difference of molecular weight is caused by aminoterminal GST tagged fusion.Expressed framework body is schematically provided in upper part of this figure.

Figure 21 display is by people isoQC (YSShisoQCI55NC351AC-His; Relatively Figure 17), GST-hisoQC and people QC transforms the specificity constant of dipeptides surrogate, dipeptides and oligopeptides.The specificity of GST-hisoQC is minimum, follows by YSShisoQCI55NC351AC-His.The people QC showing higher overall enzymic activity shows the highest specificity.

Figure 22 shows the pH-dependency of the katalysis studied with the people isoQC (hisoQC) expressed in yeast and people QC (hQC).These two kinds of protein are all presented at the optimal pH between pH7 and 8.Matched curve is based on three groups that dissociate affecting katalysis, and one of them is positioned on acid pH, and two are positioned on alkaline pH.

Figure 23 display is to the analysis transforming L-glutamic acid, and wherein said L-glutamic acid is present in the aminoterminal of amyloid-beta related peptides A β (3-11).Use Maldi-Tof mass spectroscopy to carry out this analysis, substrate and product differ and reach about 18Da on the molecular mass/charge ratio of single charged molecule, and this is the quality of the water of release.In both cases, identical protein concn is present in sample, and this clearly illustrates that people isoQC also transforms aminoterminal L-glutamic acid, but slower than people QC.

Figure 24 shows the use mouse QC (mQC, SEQIDNO:79) of real-time PCR analysis and the tissue distribution of isozyme misoQC (SEQIDNO:17) thereof.These two kinds of enzymes are all expressed in the organ of inspection.But compared with peripheral organ, the expression level of mQC is higher in brain.On the contrary, misoQC with more similar horizontal expression, represents that misoQC is a kind of extensive existence " house keeper " albumen at the whole Organ and tissues checked.

Figure 25 shows metal chelate compound 1,10-phenanthroline (circle) and the time m-dependency of EDTA (rectangle) to people isoQC (hisoQC) suppresses.Remaining hisoQC activity is directly measured afterwards in 30 DEG C of preincubation 15 minutes (open symbols) afterwards or by corresponding reagent and hisoQC in interpolation corresponding reagent (filled symbols).

Figure 26 is presented at HEK293 cells pcDNA and natural enzyme hisoQC (MetI, SEQIDNO:11) biochemical analysis, to the Subcellular Localization of QC activity after hisoQC (MetII, SEQIDNO:12) and hQC (SEQIDNO:10).(A) specific activity of unit μm ole/ minute/g in described cell.(B) absolute activity of unit nM/ minute.(C) FLAG-epi-position (anti-DYKDDDDK-antibody is detected in applying, CellSignaling), 65kDa human mitochondrion albumen (anti-human mitochondrial, Chemicon) or after the specific antibody of human sialic transferase ST1GAL3 (Abnova) carries out western blot analysis, compared with the contrast (pcDNA) of carrier transfection, h-isoQC (MetI, SEQIDNO:11), h-isoQC (MetII, SEQIDNO:12) and have hQC (SEQIDNO:10) expression in HEK293 of carboxyl terminal FLAG label.

The Subcellular Localization of people isoQC (hisoQC) signal sequence (27A) the methionine(Met) I-Serine 53 that Figure 27 display and EGFP (Isosorbide-5-Nitrae) merge and (27B) methionine(Met) II-Serine 53.Anti-mannosidase II antibody is by Golgi complex dyeing (2) and use detects the antibody of human mitochondrion 65kDa albumen by plastosome dyeing (5).Location is superposed shown (3,6) by EGFP fluorescence and RedX fluorescence altogether.

Figure 28 display and people's glycosyltransferase: α-N-acetylgalactosamine α-2,6-sialytransferase 1 (ST6GalNAC1; E.C.2.4.99.3); β-Isosorbide-5-Nitrae-galactosyltransferase 1 (b4Gal-T1, E.C.2.4.1.-); Galactoside 3 (4)-L-fucose based transferase (FucT-III; And glycoprotein-fucosido galactoside alpha-N-acetylgalactosaminyltransferase (NAGAT E.C.2.4.1.65), E.C.2.4.1.40) open sequence is compared, the structural domain structure of people isoQC (hisoQC) and mouse isoQC (misoQC).Amino acid whosely to compile under each row.Cytosolic part adds shade, transbilayer helix be black and in chamber part with white displays.

The quantification of Figure 29 display to people isoQC (QPCTL) mRNA in different carcinoma clone.QPCTL is expressed and is normalized to 50ng total serum IgE.Black rod in box represents corresponding median.

The quantification of Figure 30 display to people isoQC (QPCTL) mrna expression in different melanoma cell series.QPCTL is expressed and is normalized to 50ng total serum IgE.

Figure 31 is presented at the quantification of people isoQC (QPCTL) mRNA in the sample of soft tissue cancer, cancer of the stomach and the thyroid carcinoma being derived from different patient.QPCTL is expressed and is normalized to 50ng total serum IgE.Black rod in box represents corresponding median.

Figure 32 display in different cancer of the stomach for the quantification of people isoQC (QPCTL) mrna expression of its differentiation phase.QPCTL is expressed and is normalized to 50ng total serum IgE.Black rod in box represents corresponding median.

The quantification of Figure 33 display to people QC (QPCT) mrna expression in different thyroid carcinoma.QPCT is expressed and is normalized to 50ng total serum IgE.Black rod in box represents corresponding median.(FTC: thyroid follicular cancer; PTC: papillary thyroid carcinoma; UTC: undifferentiated thyroid carcinoma).

Figure 34 shows the comparison of people isoQC (QPCTL) mrna expression in different thyroid carcinoma.QPCTL to be expressed and is normalized to 50ng total serum IgE. the black rod in box represents corresponding median.(FTC: thyroid follicular cancer; PTC: papillary thyroid carcinoma; UTC: undifferentiated thyroid carcinoma).

Figure 35 shows the impact of different stimulated thing on the mrna expression of people QC (QPCT), people isoQC (QPCTL) and CCL2 in HEK293 cell.The amount of transcript is described relative to basal expression during non-stimulated thing.X-axis describes stimulator concentration used.

Figure 36 shows the impact of different stimulated thing on the mrna expression of people QC (QPCT), people isoQC (QPCTL) and CCL2 in FTC-133 cell.The amount of transcript is described relative to basal expression time non-stimulated.X-axis describes stimulator concentration used.

Figure 37 shows the impact of different stimulated thing on the mrna expression of people QC (QPCT), people isoQC (QPCTL) and CCL2 in THP-1 cell.The amount of transcript is described relative to basal expression time non-stimulated.X-axis describes stimulator concentration used.

Figure 38 shows the impact of different stimulated thing on the mrna expression of people QC (QPCT), CCL2, CCL7, CCL8 and CCL13 in THP-1 cell.The amount of transcript is described relative to basal expression time non-stimulated.X-axis describes stimulator concentration used.

Figure 39 shows the impact of hypoxemia on the mRNA level in-site of people QC (QPCT), people isoQC (QPCTL) and HIF1 α in HEK293 (A), FTC-133 (b) and THP-1 (C).

Sequence list

SEQ ID NO	Describe
		1	People QC, nucleic acid
2	People isoQC MetI, nucleic acid
		3	People isoQC MetII, nucleic acid
4	Cynomolgus monkey QPCTL, nucleic acid
		5	Macaque QPCTL, nucleic acid

6	Domesticated dog QPCTL, nucleic acid
		7	Rat QPCTL, nucleic acid
8	M_musculus QPCTL, nucleic acid
		9	Ox QPCTL, nucleic acid
10	People QC, protein
		11	People isoQC MetI, protein
12	People isoQC Met II, protein
		13	Cynomolgus monkey QPCTL, protein
14	Macaque QPCTL, protein
		15	Domesticated dog QPCTL, protein
16	Rat QPCTL, protein
		17	M_musculus QPCTL, protein
18	Ox QPCTL, protein
		19	People isoQC splicing form 1, nucleic acid
20	People isoQC splicing form 2, nucleic acid
		21	People isoQC splicing form 1, protein
22	People isoQC splicing form 2, protein
		23	Amyloid beta peptide (A β) (1-42)
24	Aβ(1-40)
		25	Aβ(3-42)
26	Aβ(3-40)
		27	Aβ(11-42)
28	Aβ(11-40)
		29	pGlu ³-Aβ(3-42)
30	pGlu ³-Aβ(3-40)
		31	pGlu ³-Aβ(11-42)
32	pGlu ³-Aβ(11-40)
		33	ABri
34	ADan
		35	Tert-Amyloxycarbonyltetragastrin 17

36	Tert-Amyloxycarbonyltetragastrin 34
		37	pGlu-Abri
38	pGlu-ADan
		39	PGlu-tert-Amyloxycarbonyltetragastrin 17
40	PGlu-tert-Amyloxycarbonyltetragastrin 34
		41	Neurotensin
42	GnRH
		43	CCL16
44	CCL8
		45	CCL2
46	CCL18
		47	CXXXC chemotactic molecule
48	CCL7
		49	Appetite peptide A (OrexinA)
50	Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2
		51	QYNAD
52	pGlu-YNAD
		53	For the people isoQC forward primer of cell line selection
54	For the people isoQC reverse primer of cell line selection
		55	For separating of the forward primer of people isoQC
56	For separating of the reverse primer of people isoQC
		57	For the forward primer of the isoQC that clones people (isoform Met I) to carrier pEGFP-N3
58	For the forward primer of the isoQC that clones people (isoform Met II) to carrier pEGFP-N3
		59	For the isoQC that clones people (isoform Met I and the Met II) reverse primer to carrier pEGFP-N3 to carrier pEGFP-N3
60	For the forward primer of isoQC to carrier pET41a of cloning people
		61	For the reverse primer of isoQC to carrier pET41a of cloning people
62	For the forward primer that isoQC to the carrier pPICZ α A that clones people is with carboxyl terminal histidine-tagged
		63	For the forward primer that isoQC to the carrier pPICZ α A that clones people is with aminoterminal histidine-tagged
64	For the reverse primer that isoQC to the carrier pPICZ α A that clones people is with aminoterminal histidine-tagged

65	For the forward primer of real-time PCR analysis isoQC
		66	For the reverse primer that isoQC to the carrier pPICZ α A that clones people is with carboxyl terminal histidine-tagged
67	For the reverse primer of real-time PCR analysis isoQC
		68	For cloning the forward primer of mouse isoQC cDNA
69	For cloning the reverse primer of mouse isoQC cDNA
		70	For cloning the forward primer of mouse isoQC cDNA
71	For the forward primer of real-time PCR analysis mouse QC
		72	For the reverse primer of real-time PCR analysis mouse QC
73	For the forward primer of real-time PCR analysis mouse QC
		74	For the reverse primer of real-time PCR analysis mouse QC
75	For the forward primer of site-directed mutagenesis hisoQC I55N
		76	For the reverse primer of site-directed mutagenesis hisoQC I55N
77	For the forward primer of site-directed mutagenesis hisoQC C351A
		78	For the reverse primer of site-directed mutagenesis hisoQC C351A
79	Mouse glutaminyl cyclase protein
		80	Streptomyces griseus SGAP
81	Vibrio proteolyticus VpAP
		82	For inserting the forward primer of natural hQC to pcDNA 3.1
83	For inserting the reverse primer of natural hQC to pcDNA3.1
		84	The hisoQC of terminator codon is comprised to insert the reverse primer in pcDNA 3.1 for increasing
85	For the forward primer of the EGFP that increases
		86	For the reverse primer of the EGFP that increases
87	For the hisoQC amino terminal sequence that increases with the reverse primer merged with EGFP
		88	For the hQC C-FLAG that increases to insert the reverse primer of pcDNA3.1
89	For the hisoQC C-FLAG that increases to insert the reverse primer of pcDNA 3.1

Detailed Description Of The Invention

According to aspects of the present invention, there is provided SEQIDNO:2-9,19 and 20 separated nucleic acid sequence (polynucleotide), its coding has the mature polypeptide of the derivation aminoacid sequence of QPCTL (SEQIDNO:11-18,21 and 22) from Different Origin.

According to the present invention preferably SEQIDNO:2 and 3,19 and 20 separated nucleic acid sequence (polynucleotide), its coding has the mature polypeptide of the derivation aminoacid sequence of QPCTL (SEQIDNO:11 and 12,21 and 22) from people.

According to the separated nucleic acid sequence (polynucleotide) of the present invention more preferably SEQIDNO:2 and 3, its coding has the mature polypeptide of the derivation aminoacid sequence of the people QPCTL of SEQIDNO:11 and 12.

According to the separated nucleic acid sequence (polynucleotide) of the present invention and then more preferably SEQIDNO:19 and 20, its coding has the mature polypeptide of the derivation aminoacid sequence of the people QPCTL variable sheer form of SEQIDNO:21 and 22.

According to the separated nucleic acid sequence (polynucleotide) of the present invention most preferably SEQIDNO:2, its coding has the mature polypeptide of the derivation aminoacid sequence of the people QPCTL of SEQIDNO:11.

According to the separated nucleic acid sequence (polynucleotide) of the present invention and then most preferably SEQIDNO:3, its coding has the mature polypeptide of the derivation aminoacid sequence of the people QPCTL of SEQIDNO:12.

Foregoing embodiments and preferable be applicable to QPCTL nucleic acid and QPCTL albumen and any want use, diagnosis, treatment, screening method, effector, inhibitor and other purposes of the present invention and method.

Be used in NCBI ( http:// www.ncbi.nlm.nih.gov/BLAST/) on Nucleotide BLAST, utilize people QC as template, find polynucleotide of the present invention by similarity searching.This search causes the presumption QPCTL finding to be positioned on karyomit(e) 19, and its coding region is in 19q13.32 region.Based on this search, devise the primer (table 4) for using cell line selection people isoQC.The separation cDNA of people QPCTL contains the open reading-frame (ORF) of the protein of coding 382 amino acid lengths, and wherein said open reading-frame (ORF) is relevant to people QC, the sequence thereto of performance 45.24% and 71.98% similarity.The different bioinformatics of applied forcasting Subcellular Localization ( www.expasy.ch) do not produce reliable results.According to described predictor, prediction is diverted golgi body or plastosome.

In the M28 family member of people QPCTL and metallopeptidase ClanMH, the amino acid alignment result of other member shows people QPCTL albumen and has and the overall sequence homology of people and mouse QC (Fig. 1) and bacterium aminopeptidase (Fig. 3) and structural homology.The database search of extra people QPCTL genes involved is disclosed and there is rodent, monkey, ox and dog QPCTL.The comparison result of these sequences and described novel people QPCTL shows them and shows the huge homology with its mankind's counterpart.The zinc complexing residue of people QC (Asp-Glu-His) guards (Fig. 2) in from the QPCTL of Different Origin.

People isoQC gene contains at least 8 exons.The sequence of encoding human isoQC protein is positioned at the 1st to the 7th exon.People isoQC is positioned No. 19 chromosome position 19q13.32 place.Cell line selection method for people isoQC discloses from liver (Hep-G2, hepatocellular carcinoma), skin (HaCaT, keratinocyte) and neuronal tissue (LN405, astrocytoma; SH-SY5Y, neuroblastoma) cell source (Fig. 6) in transcript.

The functional expression of the QPCTL-cDNA of separation is tested in several expressive host.The pichia pastoris phaff (P.pastoris) being successfully applied to people QC expresses the protein not having generation to have enzymic activity.Expression in mammalian cell causes activity being detected, but expression level is extremely low.Therefore, the protein of enzymic activity can not be separated with the knowledge of technician.Only use in the intestinal bacteria (E.coli) unconventional expression condition (existence 37 DEG C in 1% glucose express 4 hours, use 20 μMs of IPTG abduction deliverings) express GST-QPCTL fusion rotein after be just separated to the protein of enzymic activity.Described expression condition produces low expression level in intestinal bacteria, and it is required that this is that described peptide chain generating function folds.

In another embodiment, the present invention relates to QPCTL knock-out animal.Described animal is preferably rat or mouse.Knock-out mice is a kind of important tool analysing in depth the use in QPCTL gene function.

Polynucleotide of the present invention can be rna form or DNA form; DNA should be understood to comprise cDNA, genomic dna and synthetic DNA.Described DNA can be double-strand or strand, and if in strand, then this DNA can be coding strand or non-coding (antisense) chain.The encoding sequence of encoding mature polypeptide can be identical with the encoding sequence shown in SEQIDNO:2-9, or it can be different from the encoding sequence of coding same mature polypeptide because of the redundancy of genetic codon or degeneracy or single nucleotide polymorphism.Such as, described encoding sequence also can be the rna transcription thing comprising the arbitrary complete length of SEQIDNO:11-18.

The polynucleotide of the maturation protein of coding SEQIDNO:2-9 can include, but are not limited to the encoding sequence of described maturation protein itself; The encoding sequence of the additional additional coding sequence of described mature polypeptide (as leading or secretion sequence or proprotein sequence) and as described in maturation protein encoding sequence (with optionally additional coding sequence) additional non-coding sequence (as intron or as described in 5 ' and/or the 3 ' non-coding sequence of encoding sequence of maturation protein).

Therefore, term " polynucleotide of coded polypeptide " or term " nucleic acid of coded polypeptide " should be understood to the polynucleotide of the encoding sequence only comprising described maturation protein or nucleic acid and comprise polynucleotide or the nucleic acid of extra coding and/or non-coding sequence.Term " polynucleotide " and " nucleic acid " use interchangeably.

The present invention also comprises such polynucleotide, and the encoding sequence of wherein said maturation protein can merge identical open reading-frame (ORF) with auxiliary expression polypeptide and from the polynucleotide sequence of host cell secretes; Such as, the leader sequence played a role as the secretion sequence controlled from cell transport polypeptide can merge in such a way.The polypeptide with this leader sequence is called proteinogen or preproprotein and can excises this leader sequence to form the mature form of this protein by host cell.These polynucleotide can have 5 ' elongated area, thus such proteinogen of encoding, it is that maturation protein is added to N-terminal additional amino acid residue.The expression product with former sequence (prosequence) like this is called proteinogen, and it is the inactive form of described maturation protein; But, once this former sequence is cleaved, then leave activated maturation protein.Therefore, such as, polynucleotide of the present invention encoding mature albumen or coding can have the protein of former sequence or have the protein of former sequence and presequence (leader sequence).

Polynucleotide of the present invention also can have and the encoding sequence allowing the flag sequence of purifying polypeptide of the present invention to merge to meet open reading-frame (ORF) mode.When using mammiferous host such as COS-1 cell, described flag sequence can be polyhistidine tag, hemagglutinin (HA) tag, c-myc label or V5 label.

HA label corresponds to from the protein derived epi-position (Wilson of influenza virus hemagglutinin, I. etc., Cell, 37:767 (1984)) and c-myc label can be epi-position (Evans from people Myc albumen, G.I. etc., Mol.Cell.Biol.5:3610-3616 (1985)).

Term " gene " means the sections participating in the DNA producing polypeptide chain; It is included in the intervening sequence (intron) between region before and after coding region (leader and trail district) and each encoding segments (exon).

Term " significant sequence homology " means at least 25%, preferably at least 40% amino-acid residue guard, and the non-conservative residue of at least 40% is preservative replacement.

The fragment of full-length gene of the present invention can be used as the hybridization probe for cDNA library, has remarkable sequence homology and other cDNA of the protein with similar biological activity or function of encoding to be separated full-length cDNA with being separated with this gene.For the application's object, similar biologic activity or function mean to be defined as the aminoterminal glutamine from peptide, protein, hormone or other substrate of QC activity and EC activity or the ability of L-glutamic acid formation pyroglutamic acid ester respectively.Such probe has at least 14 bases (at least 14 continuous nucleotides from one of SEQIDNO:2-9), preferably at least 30 bases, and this probe can containing such as 50 or more bases.The probe of preferred SEQIDNO:53-61.This probe also can be used for identifying that the cDNA corresponding with total length transcript and/or the genomic clone containing complete genome (comprising regulatory region and promoter region, exon and intron) clones.Have with the labeled oligonucleotide of the sequence of the complementary of gene of the present invention for screening the library of people cDNA, genomic dna or mRNA or originating or the similar library of animal, to determine the library constructs with described probe hybridization from other.As an example, known dna sequence can be used to carry out synthetic oligonucleotide probe, the latter is used for being separated the coding region of goal gene subsequently in library screening.

The present invention's imagination also provides the polynucleotide with hereinbefore sequence hybridization, have at least 70% between wherein said sequence, preferably at least 90% and more preferably at least 95% homogeny or similarity, and therefore described sequence is encoded and is had the protein of similar biological activity.In addition, as known in the art, when aminoacid sequence contains the identical or Conservative amino acid substitute of each residue in for sequence, between two polypeptide, there is " similarity ".Homogeny and similarity can use sequence analysis software (such as PBIL ( bioinformatiqueLyonnais) http:// npsa-pbil.ibcp.fr) on ClustalW) measure.The present invention is provided in these type of polynucleotide with hereinbefore multi-nucleotide hybrid under stringency especially.As used herein, term " stringency " means to allow to hybridize in case depositing at least about 70% homogeny between polynucleotide sequence and the polynucleotide sequence of SEQIDNO:2-9.

Appropriate stringency conditions by the concentration of salt and methane amide in such as prehybridization solution and hybridization solution or can be defined by hybridization temperature, and is well known in the art.Especially, severity can by reducing salt concn, by increasing concentration of forma and/or improving by raising hybridization temperature.

Such as, the hybridization under High stringency conditions can use about 50% methane amide on about 37 DEG C-42 DEG C, and the hybridization under reduction stringency can use about 35%-25% methane amide methane amide on about 30 DEG C-35 DEG C.Specified conditions for hybridizing under High stringency conditions combinationally use 42 DEG C, and 50% methane amide, 5 × SSPE, 0.3%SDS and 200 μ g/ml shear and sex change salmon sperm DNA.For reducing the hybridization under severity, can use and similar condition mentioned above in the temperature 35 DEG C reduced in 35% methane amide.The temperature range corresponding with specific Stringency levels can be reduced further than also regulating temperature accordingly by the purine/pyrimidine calculating object nucleic acid.Well known change in above-mentioned scope and condition.Preferably, hybridization only should have at least 95% between sequence and more preferably at least 97% homogeny time just occur.With the polypeptide that the polynucleotide encoding of the multi-nucleotide hybrid mentioned above in preferred embodiment is such, it shows the substantially the same biological function of the maturation protein coded with one of cDNA of SEQIDNO:2-9 or activity.

As mentioned, suitable polynucleotide probes can have at least 14 bases, preferably 30 bases and more preferably at least 50 bases, and it is as discussed herein above, will with multi-nucleotide hybrid of the present invention, wherein said polynucleotide of the present invention and described probe have homogeny and can to retain or can not retentive activity.Such as, these type of polynucleotide can be used as respectively with the probe of the multi-nucleotide hybrid of EQIDNOS:2-9, such as, for reclaiming this polynucleotide, or be used as diagnostic probe or be used as PCR primer.Therefore, the present invention includes such polynucleotide, the polypeptide of its polynucleotide with the polypeptide of the SEQIDNO:11-18 that to encode respectively or its fragment and therewith class nucleotide coding have at least 70% homogeny, preferred at least 90% homogeny, wherein said fragment preferably has at least 30 bases and more preferably at least 50 bases.

As known in the art, genetic codon is redundancy, namely some amino acid is encoded by more than one nucleotide triplet (codon), and the present invention includes and use the codon not identical with the codon that specifically exemplifies in this paper sequence to encode those polynucleotide sequences of same amino acid.This polynucleotide sequence is called " equivalence " polynucleotide sequence in this article.The present invention also comprises the variant of above-described polynucleotide herein, and described variant is encoded such fragment, as having the maturation protein of one of the polypeptide of the derivation aminoacid sequence of any one of SEQIDNO:11-18, the part or all of of sum analogous to general Dedekind sum.The variant form of described polynucleotide can be the natural existence allelic variant of these polynucleotide or the non-natural existence variant of these polynucleotide.Such as, the variant in described nucleic acid can be the amino acid codes sequence difference only produced because of genetic codon degeneracy, maybe can there is deletion mutants, displacement variant and interpolation or insert variant.As known in the art, allelic variant is the variant form of polynucleotide sequence, and it can have the displacement of one or more Nucleotide of the biological function substantially not changing coded polypeptide, disappearance or interpolation.

The present invention also comprises the polypeptide of the derivation aminoacid sequence with SEQIDNO:11-18, and the fragment of this type of polypeptide, sum analogous to general Dedekind sum.When referring to the polypeptide of SEQIDNO:11-18, term " fragment ", " derivative " mean substantially to retain the biological function identical with this type of polypeptide or the polypeptide of activity with " analogue ".Analogue can such as comprise such proteinogen, and this proteinogen can activate because of this proprotein portion cracking to produce active maturation protein.Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides or improvement on synthesis; But they are glycosylation or non-glycosylated recombinant polypeptide preferably.

The fragment of the polypeptide that SEQIDNO:11-18 is arbitrary, derivative or analogue can be (i) such a kind of fragments, derivative or analogue, wherein one or more amino-acid residues replace with conservative or nonconserved amino acid residues (preferred conservative amino acid residues) and the amino-acid residue of this displacement can be or can not be the amino-acid residue of genetic codon coding, or a kind of fragment that (ii) is such, derivative or analogue, wherein one or more amino-acid residues comprise substituting group, or a kind of fragment that (iii) is such, derivative or analogue, wherein extra amino acid and maturation protein merge, as leader sequence or secretion sequence or the sequence for mature polypeptide machine or proprotein sequence as described in purifying.This type of fragment, derivative and analogue are those skilled in the art can be provided based on teachings herein.

Polypeptide of the present invention should be under the form be separated with polynucleotide, and preferably they is purified to substantially homogeneous or pure.Substantially the homogeneous purity meant at least about 85%.

Term " separation " is used for meaning material and shifts out from its initial environment (if such as this material is naturally occurring, then its natural surroundings).Such as, in the animal lived, the natural existence polynucleotide that exist or polypeptide are not considered as being separated, not excessive identical polynucleotide or polypeptide substantially with natural system in all compossibility material separates time, be then be separated depending on it.For DNA; this term comprises such as such recombinant DNA; wherein said recombinant DNA is incorporated to carrier, self-replicating character grain or virus or prokaryotic gene group or eukaryotic gene group, or as the independent molecule existence molecule (such as digesting the cDNA of generation or genomic fragment or cDNA fragment by polymerase chain reaction (PCR) or restriction endonuclease) independent of other sequence.This term also comprises the recombinant DNA of the part of the heterozygous genes as encoding additional polypeptide sequence (as fusion rotein).This term also comprises such recombinant DNA, and it comprises a part for Nucleotide shown in SEQIDNO:2-9, the alternative splice variants of described code segment QPCTL.Multiple alternative splice variants is exemplified in SEQIDNO:19-22.

Polypeptide of the present invention comprise SEQIDNO:11-18 arbitrary polypeptide (especially maturation protein) and with one of polypeptide of SEQIDNO:11-18 have at least 75% similarity (such as preferably at least 50% and more preferably at least 70% homogeny) polypeptide, more preferably there is with one of polypeptide of SEQIDNO:11-18 at least 85% similarity (such as preferably at least 70% homogeny) and most preferably have with arbitrary polypeptide of SEQIDNO:11-18 at least 95% similarity (such as preferably at least 90% homogeny).In addition, polypeptide of the present invention preferably should comprise the clear and definite part of this type of polypeptide, and described clear and definite part contains at least 30 amino acid and more preferably at least 50 amino acid whose sequences.

The fragment of polypeptide of the present invention or part can be used as intermediate to produce corresponding full-length polypeptide by method of peptide synthesis.Fragment or the part of polynucleotide of the present invention also can be used for synthesizing total length polynucleotide of the present invention.

The present invention also comprises the carrier containing these type of polynucleotide, carries out genetically engineered host cell and use these carriers and host cell to produce polypeptide by recombinant technology with described carrier.The such carrier of host cell carries out genetically engineered (transduce or transform or transfection), and wherein said carrier can be such as cloning vector or expression vector.Described carrier can be the forms such as plasmid, virus particle, phage.The host cell of through engineering approaches can be cultivate in conventional nutrient culture, and wherein said conventional nutrient culture improves for activating promotor, selecting transformant or amplification gene of the present invention.As technician knows, culture condition as temperature, pH etc. be with selected for the host cell of expressing those culture condition normally used.

Polynucleotide of the present invention may be used for producing polypeptide by recombinant technology.Therefore, such as, described polynucleotide can be comprised in arbitrary carrier of multiple expression vectors of express polypeptide.Examples of such carriers comprises karyomit(e), non-chromosome and synthetic DNA sequence dna, the derivative of such as SV40; Bacterial plasmid; Phage DNA; Baculovirus; Yeast plasmid; Carrier derivative from the combination of plasmid and phage DNA; Viral DNA is as vaccinia virus, adenovirus, bird pox virus and Pseudorabies virus.But, other carrier any can be used, as long as it is reproducible and have vigor in host.

By multiple method, suitable DNA sequence dna can be inserted described carrier.Generally speaking, described DNA sequence dna inserts suitable restriction endonuclease site by method well known in the art, and wherein said method is in the limit of power of those skilled in the art.

DNA sequence dna in expression vector is effectively connected with the suitable expression control sequenc (promotor) instructing mRNA to synthesize.As the representative example of this type of promotor, can mention: the P.sub.L promotor of LTR or SV40 promotor, intestinal bacteria lac or trp, phageλ and known other promotor that controlling gene is expressed in prokaryotic cell prokaryocyte or eukaryotic cell or its virus.

Described expression vector also should containing for the ribosome bind site of translation initiation and transcription terminator.This carrier also can comprise for the appropriate sequences of expressing that increases.In addition, this expression vector preferably contains one or more selectable marker genes to provide the phenotypic attributes selecting transformed host cell, eukaryotic cell is cultivated, marker gene is such as Tetrahydrofolate dehydrogenase or neomycin resistance, or in intestinal bacteria, such as, be tetracyclin resistance or amicillin resistance.

Carrier containing, for example the above-described suitable DNA sequence dna of this paper and appropriate promoters or control sequence can be used for transforming appropriate host to make this host expresses protein.As the representative example of suitable host, can should be mentioned that: bacterial cell is as intestinal bacteria, streptomycete (Streptomyces), Salmonella typhimurium (Salmonellatyphimurium); Fungal cell is as yeast; Insect cell, as fruit bat (Drosophila) S2 and noctuid (Spodoptera) Sf9; Zooblast is as CHO, COS or Bowes melanoma; Adenovirus; Vegetable cell etc. the selection of suitable host is in the limit of power of those skilled in the art because of teachings herein.

As from CLONTECH95/96 catalogue 215-216 page, CLONTECH, 1020EastMeadowCircle, PaloAlto, Calif.94303 are apparent, the synthetic generation of well known nucleotide sequence.Therefore, the present invention also comprises the expression vector for generation of present protein.Present invention includes the recombinant precursor of the one or more sequences comprised as above-outlined.This construct can comprise wherein to insert the carrier of sequence of the present invention forward or backwards, as plasmid or virus vector.This embodiment preferred in, described construct also comprises the adjustment sequence be effectively connected with sequence of the present invention, and described adjustment sequence comprises such as promotor.Numerous suitable carrier and promotor are well known by persons skilled in the art and can business obtain.There is provided following carrier by way of example: bacteria carrier: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, psiX174, pbluescriptSK, pbsks, pNH8A, pNH16a, pNHI8A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR54 and pRIT5 (Pharmacia); And eukaryotic vector: pWLNEO, pSV2CAT, pOG44, pXTI, pSG (Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia).But, can use any other suitable plasmids or carrier, as long as it is reproducible and have vigor in host.

Promoter region can be any required gene being selected from other carrier using CAT (E.C. 2.3.1.28) carrier or have selective marker.

Two kinds of appropriate carrier are pKK232-8 and pCM7.The promoters specifically mentioned comprises lacI, lacZ, T3, T7, gpt, λ P.sub.R, P.sub.L and trp.Eukaryotic promoter comprises the early stage and late promoter of CMV immediate early promoter, HSV thymidine kinase, SV40, from retroviral LTR and Mouse Metallothionein-I promotor.Appropriate carrier and promotor is selected then to be in completely in the limit of power of those skilled in the art.

Expression vector assembly generally can comprise: 1) as neomycin phosphotransferase (G418) or hygromycin B phosphotransferase (hyg) gene, 2 of selective marker) replication orgin, 3) T7 and SP6 bacteriophage promoter sequences, 4) lac operator gene sequence, 5) lactose operon repressor gene (lacIq) and 6) multiple clone site connector area.This replication orgin (oriC) can derive from pUC19 (LTI, Gaithersburg, Md.).

According to the PCR method described in Examples below 1 and 2, PCR primer is used to generate the nucleotide sequence with one of the polypeptide of the coding SEQIDNO:2-9 of appropriate restriction sites, wherein said PCR primer has and is intended to clone isoQCMetI and MetII to the restriction site EcoRI (as 5 ' primer) in carrier EGFP-N3 and SalI (as 3 ' primer), or for the site SpeI (as 5 ' primer) that clones isoQC to carrier pET41a and EcoRI (as 3 ' primer).PCR inset is carried out gel-purified and digests with consistency Restriction Enzyme.Described inset and carrier is connected according to standard scheme.

In still another embodiment, the invention provides the host cell containing above-mentioned construct.This host cell can be higher eukaryotic cell, and as mammalian cell, or lower eukaryotes cell is as yeast cell, or described host cell can be prokaryotic cell prokaryocyte, as bacterial cell.Calcium phosphate transfection method, DEAE-dextran mediated transfection method, lipofection or electroporation (Davis can be passed through, L., Dibner, M., Battey, I., BasicMethodsinMolecularBiology, (1986)) implement to import described construct to host cell.

Preferably in host cell, use this type of construct produce the gene product of being encoded by recombination sequence in a usual manner.Or polypeptide of the present invention can be produced synthetically by conventional peptide synthesizers or connects the suitable fragments of so preparation by chemistry and produce.

Maturation protein can be expressed in mammalian cell, yeast, bacterium or other cell under the control of suitable promoter.Also can use cell free translation system to utilize RNA derivative from DNA construct of the present invention to produce this proteinoid.The suitable cloning vector used with prokaryotic hosts and eucaryon host and expression vector by Sambrook, etc., MolecularCloning:ALaboratoryManual, SecondEdition, ColdSpringHarbor, N.Y., (1989) describe.

By improving higher eucaryote transcribing the DNA of code book invention polypeptide by enhancer sequence insertion vector.

Enhanser comprises the DNA cis-acting elements acting on promotor and transcribe to improve it, and its length is about 10-300bp usually.The example of enhanser is included in the SV40 enhanser of replication orgin side in late period 100-270bp, cytomegalovirus early promoter/enhanser, the many types of knurl enhanser of replication orgin side in late period and adenovirus cancers.

Usually, recombinant expression vector will comprise replication orgin and impel the selective marker of transformation of host cells (such as E. coli ampicillin resistant gene and yeast saccharomyces cerevisiae (S.cerevisiae) TRP1 gene) and derive the promotor that instructs downstream structural sequence to transcribe from cance high-expression gene.This type of promotor can from encoding glycolytic enzymes as the operon of glycerol 3-phosphate acid kinase (PGK), α-factor, acid phosphatase or heat shock protein(HSP) etc. derives.Heterologous structural sequence is with suitable way and translation initiation sequence and terminator sequence and preferably assemble with leader sequence, and wherein said leader sequence can instruct the protein secreting of translation in periplasmic space or the outer substratum of born of the same parents.Optionally, described heterologous sequence can encoded packets containing the fusion rotein of aminoterminal identification polypeptide, wherein said aminoterminal identification polypeptide give needed for feature, as stable expressed recombinant products with simplify its purifying.

The initial expression vector built for bacterium with termination signal of effective reading suitable translation is mutually positioned at together with functional promoter by the structural DNA sequence inserting encode desired proteins.

This carrier will comprise one or more phenotypic selection markers and replication orgin to guarantee maintain this carrier and provide amplification as required in host.Suitable prokaryotic hosts for transforming comprises the multiple species in intestinal bacteria, subtilis (Bacillussubtilis), Salmonella typhimurium and Rhodopseudomonas (Pseudomonas), streptomyces (Streptomyces) and Staphylococcus (Staphylococcus), but also can select other prokaryotic hosts.

Representatively property and limiting examples, for the useful expression vector of bacterial use can comprise from containing know cloning vector pBR322 (ATCC37017) genetic elements commercially available plasmid derivative selective marker and bacterial origin of replication.This type of commercialization carrier comprises, such as, and pKK223-3 (PharmaciaFineChemicals, Uppsala, Sweden) and GEM1 (PromegaBiotec, Madison, Wis., U.S.A.).These pBR322 " main chain " part combines with appropriate promoters and structural sequence to be expressed.

At conversion suitable host strain after cultivating this host strain to suitable cell density, by the promotor selected by suitable method (such as, temperature inversion or chemical induction) induction and by the time period extra for cell cultures.

Cell is generally also destroyed by physics or chemical means subsequently by collected by centrifugation, leaves gained crude extract for being further purified.

Can destroy microorganism cells used in protein expression by any facilitated method, described method comprises freeze-thaw cycle method, Ultrasonic treatment, physical disturbance method and utilizes lysis agent; These class methods are well known to those skilled in the art.

Multiple mammalian cell culture system also can be used for expressing recombinant protein.The example of mammalian expression system comprises Gluzman, the monkey kidney fibroblast COS-7 that Cell, 23:175 (1981) describe.Other clone can expressing compatible carriers comprises such as C127,3T3, CHO, HeLa and bhk cell system.Mammiferous expression vector generally comprises replication orgin, suitable promotor and enhanser and comprises ribosome bind site required arbitrarily, site of polyadenylation, donor splicing site and acceptor site, transcription termination sequence and 5 ' flank non-translated sequence.The DNA sequence dna derivative from SV40 spliceosome and site of polyadenylation can be used to provide required non transcribed genetic elements.

Polypeptide can by comprise ammonium sulfate or ethanol precipitation, acid extraction method, negatively charged ion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography method, affinity chromatography, hydroxylapatite chromatography and lectin chromatography method reclaim from recombinant cell culture thing.If described expression of polypeptides is on cell surface, then recovery can be assisted, but this not precondition.Also can reclaim the product of cracking, wherein said split product is subject to cracking after the more microscler formula of described polypeptide is expressed.As required, Protein refolding steps known in the art can be used to realize the conformation of maturation protein.High performance liquid chromatography (HPLC) may be used for final purification step.

Polypeptide of the present invention can be the natural product of purifying or be produced from protokaryon or eucaryon host (such as passing through the bacterium of cultivation, yeast, higher plant, insect or mammalian cell) by recombinant technology.According to the host used in restructuring production method, polypeptide of the present invention can be glycosylated can be maybe nonglycosylated.Polypeptide of the present invention also can comprise an initial methionine amino-acid residue.

In preferred embodiments, protein of the present invention is abstraction and purification, thus is substantially devoid of the impurity from other oroteins.Such as, protein of the present invention should account at least 80% (weight), more preferably at least 90%, even more preferably at least 95% and most preferably account at least 98% (weight) of gross protein of the gross protein existed in sample.

These protein can be as the solution form in the mixture of dimethyl sulfoxide (DMSO) (DMSO) or ethanol or suitable solvent at water, another kind of suitable solvent.

The example of solvent mixture is included in (weight) ethanol of 10% in water and 2% (weight) DMSO in water.Solution can also comprise salt, buffer reagent, chaotropic agent, washing agent, sanitas etc.Or described protein can be solid form, as lyophilized powder or crystalline solid, this form also can comprise residual solvent, salt etc.

As used herein, term " antibody " comprises polyclonal antibody, the polyclonal antibody of affinity purification, monoclonal antibody and Fab, as F (ab ') ₂with Fab ' proteolytic fragment.Also genetically engineered complete antibody or fragment is comprised, as chimeric antibody, Fv fragment, strand chain antibody etc. and synthetic hla binding peptide and polypeptide.Non-human antibody can carry out humanization by transplanting on inhuman CDR to people's framework and constant region or by being incorporated to complete non-human variable domains's (optionally by the residue of replacement exposure with non-human variable domains described in proper manners surface coverage, wherein products therefrom is " inlaying (veneered) " antibody).In some cases, humanized antibody can retain non-human residues in people's variable framework structural domain to strengthen correct combination feature.By making antibody humanization, can biological half-life be improved and the unfavorable immune response after being applied to people can be reduced in.

For generation of or select the alternatives of useful antibody herein to comprise the external lymphocyte that makes and be exposed to people isoQC protein or its peptide, and in phage or similar bearer, select antibody display libraries (such as by using people isoQC protein or the peptide of immobilization or mark).

Can be upper or obtain at the upper random library shown of bacterium (as intestinal bacteria) gene that coding has potentiality people isoQC polypeptide integrated structure domain polypeptide in phage (phage display) by screening.The nucleotide sequence of encoding such polypeptides can obtain according to numerous method well known in the art.

As those skilled in the art obviously understand, polyclonal antibody can inoculate multiple warm-blooded animal as horse, milk cow, goat, sheep, dog, chicken, rabbit, Mouse and rat and producing by employment isoQC polypeptide or its fragment.The immunogenicity of people isoQC polypeptide can by using adjuvant as aluminium adjuvant (aluminium hydroxide) or Freund's complete adjuvant or Freund's incomplete adjuvant, or surfactant such as lysolecithin, Pluronic polyvalent alcohol, polyanion, peptide, oil-emulsion, KLH or dinitrophenol are improved.Especially preferably bacille Calmette-Guerin vaccine BCG (bacilliCalmette-Guerin) and CBP (Corynebacteriumparvum) in for the adjuvant of the mankind.Polypeptide for immunity also comprises fusion polypeptide, as isoQC or its part and immunoglobulin polypeptides or the fusions with maltose binding protein.Polypeptide immunogen can be full-length molecule or its part.If described polypeptide portion is " haptens sample ", then this part can with macromolecular carrier as keyhole (worm relative) hemocyanin (KLH), and bovine serum albumin (BSA) or Toxoid,tetanus are advantageously combined or connect for immunity.Also method well known in the art generation can be used for the antibody of isoQC.The fragment that this antibody-like can include, but are not limited to polyclone, mono-clonal, chimeric and single-chain antibody, Fab fragment and be produced by Fab expression library.

Neutralizing antibody (namely block or regulate at reactive site place those antibody interactional) is especially preferred for therapeutic use.

For producing the antibody with QPCTL specific binding, binding proteins or peptide, can screen the library of single-chain antibody, Fab fragment, other antibody fragment, non-antibody protein structural domain or peptide.Described library can use phage, other recombinant DNA method or method of peptide synthesis produce NatureBiotechnology14:309-314 (1966) such as () Vaughan, T.J..This type of library uses usually is intended to identify that display is screened with the well known method of the sequence of QPCTL specific binding.

Preferably, the oligopeptides of anti-QPCTL antibody, peptide or fragment is used for inducing to have by least about 5 amino acid and more preferably by the aminoacid sequence formed at least about 10 amino acid.Also preferably these oligopeptides, peptide or fragment are identical with a part for the aminoacid sequence of natural protein.QPCTL amino acid whose short-movie section also can merge with those short sections of another kind of protein (as KLH), and can produce the antibody for this chimeric molecule.

Monoclonal antibody for QPCTL can use knows technology preparation arbitrarily, and wherein said technology of knowing produces antibody molecule by the continuous cell line cultivated.These technology include but not limited to hybridoma technology, people B-cell hybridoma technique and EBV-hybridoma technology, but the monoclonal antibody that can preferably be produced by hybridoma.

In addition, the technology being intended to produce " chimeric antibody " can be used, as mouse antibody gene montage is had the molecule of appropriate antigen specificity and biologic activity, see the Nature312:604-608 such as Neuberger, M.S. (1984) to human immunoglobulin gene to obtain.Can improve and to use methods known in the art, QPCTL specific single-chain antibody is produced to the technology produced described by single-chain antibody.Relative specific can be had by producing from the chain reorganization effect immunoglobulin (Ig) random combinatorial libraries, but the antibody (BurtonD.R.Proc.Natl.Acad.Sci.88:11120-11123 (1991)) of different idiotype composition.

As disclosed in document, antibody also can produce Proc.Natl.Acad.Sci.86:3833-3837 (1989) such as () Orlandi, R. by producing in the body in induction of lymphocyte colony or go out the reagent of high degree of specificity combination by screening immunoglobin libraries or elutriation.

Also can produce containing the antibody fragment for the specific binding site of QPCTL.Such as, this type of fragment includes, but are not limited to the F (ab ') that produced by pepsin digested antibody molecule ₂fragment and by reduction F (ab ') ₂the disulphide bridges of fragment and the Fab fragment produced.Or, Fab expression library can be built and to come rapidly and easily qualification has required specific Monoclonal Fab fragments Science254:1275-1281 (1989) such as () Huse, W.D..

Panimmunity assay method can be used identify and there is required specific antibody.Well known use has establishes the competitive binding assay method of specific polyclone or monoclonal antibody or numerous schemes of immunoradiometric assay.This type of immunoassay generally comprises the mixture measured between QPCTL and its specific antibody and is formed.Preferred utilization is to the immunoassay based on dibit point monoclonal antibody of the monoclonal antibody that two non-interfering QPCTL epi-positions respond.But also can use competitive binding assay method.

As comparatively early mentioned, QPCTL can be used in disease treatment.

The pharmaceutical composition being applicable to this aspect of the invention comprises such composition, wherein comprises activeconstituents to realize the expection object significant quantity relevant to one of described disease.In the limit of power that the determination for the treatment of significant quantity is in those skilled in the art completely and can at first at cell culture assays (as in cancer cells cultivation assay method) or set up in animal model, described animal model is mouse, rat, rabbit, dog or pig normally.Animal model also can be used for the application concentration scope determining to be suitable for and approach, and described information is commonly used to determine to be used in the application dosage in the mankind and approach subsequently.

Treatment significant quantity refers to that activeconstituents such as QPCTL or its fragment, DPRP antibody or QPCTL agonist, antagonist or inhibitor can alleviate the amount of the concrete symptom of disease or situation.Such as, amount to be administered can at aminoterminal Glu or Gln with this target substrate of effective cyclisation during expection target substrate contact.Treatment effect and toxicity can be determined in cell cultures or with laboratory animal similarly by standard pharmaceutical procedures, as passed through to calculate ED50 (in 50% colony the effective dosage of therapeutic) or LD50 (dosage lethal in 50% colony) statistic.The dose ratio of poisonous effect/response to treatment is therapeutic index, and it can be expressed as LD50/ED50 ratio.The pharmaceutical composition of the therapeutic index that preferred display is large.The data obtained from cell culture assays and zooscopy use in the dosage range drawing up people's purposes.Dosage contained in such composition is preferably within the scope of such circulation composition, and it comprises the small or avirulent ED50 of toxicity.Described dosage changes according to formulation used, patient sensitivity and route of administration within the scope of this.

By Medical practitioners, precise dosage generally can consider that the factor relevant to the object requiring to treat is determined, adjustment dosage and application process are to provide the active part of enough levels and to maintain the effect wanted simultaneously.Factor to be considered comprises the seriousness of morbid state, object general health situation, subject age, body weight and sex, diet, time of application and frequency, medication combined, reaction sensitivity and the tolerance/response to treatment.According to half life and the clearance rate of concrete preparation, depot drug product composition can be used every 2 weeks every 3-4 day or even.

Another aspect of the present invention provides such polynucleotide molecule, and it has with the polynucleotide mRNA transcript of SEQIDNO:2-9 is the sequence of antisense.Use antisense polynucleotides molecule can block by the generation of the QPCTL coded by said gene protein of SEQIDNO:2-9.Known in the art for the preparation of antisense polynucleotides molecule and the technology using this quasi-molecule.Such as, antisense polynucleotides molecule can be wrapping in liposome so that cytogamy.

Especially, be expressed as this gene latent effect in the pathology of hereinafter described disease of the QPCTL gene of SEQIDNO:2-9 in brain, prostate gland, lung, heart, liver, spleen and renal tissue is produced evidence.Therefore, in yet another aspect, the present invention relates to the diagnostic assay method for detecting active to suitable QPCTL or that expression level is relevant disease.The antibody of specific binding QPCTL may be used for diagnosis and is expressed as the illness of feature with QPCTL or uses in the assay method for monitoring patient, wherein said patient QPCTL or treat with the agonist of QPCTL or antagonist (inhibitor).Can prepare according to mode identical to those antibody described by therapeutant above for diagnostic purpose antibody.Comprising for QPCTL diagnostic assay method utilizes described antibody and marker to come in human body liquid or the method for QPCTL in the extract of cell or tissue.Described antibody in modification or can use when not modifying, and they can be marked by covalently or non-covalently being connected with reporter molecules.Type known in the art reporter molecules widely.Also can use modified thus the restructuring QPCTL albumen of catalytically inactive as dominant negative sense inhibitor.This type of is modified to comprise and such as suddenlys change to reactive site.

Ability becomes known for the multiple method measuring QPCTL, comprises ELISA, RIA and FACS, and described method provides QPCTL expression level that is that diagnosis changes or exception.By setting up taking from the body fluid of normal mammalian object, preferably people or cell extract the normal value or standard value that QPCTL expresses being suitable for merging with anti-QPCTL antibody under the condition forming mixture.Method for detecting QPCTL in biological sample will comprise step a) provides biological sample; B) this biological sample and anti-QPCTL antibody are being suitable for merging under the condition forming mixture between QPCTL and this antibody; With c) mixture examined between QPCTL and this antibody is formed, and thus determines the existence of QPCTL in this biological sample.

Subsequently can by multiple method, the amount that formed preferably by the quantitative mixture of photometry.Express in object, contrast and compare with described standard value from the QPCTL amount in the disease sample of biopsy.Deviation between standard value and object value sets up the parameter for diagnosing the illness.

In another embodiment of the invention, for diagnostic purpose, use the polynucleotide of coding QPCTL, wherein said polynucleotide can comprise oligonucleotide sequence, complementary RNA and DNA molecular and PNA.These polynucleotide can be used for detect and quantitative biopsy in genetic expression, in described biopsy, the expression of QPCTL can be relevant to one of described disease.Described diagnostic assay method can be used for distinguishing QPCTL and do not exist, exists and overexpression be used for adjustment to QPCTL level during monitor therapy intervention.In addition, the pharmacogenomics of QPCTL gene, single nucleotide polymorphism (SNP) can be used to analyze the method for suddenling change as screening, and wherein said sudden change indicates disease prognosis or replys the improvement of medicine.

QPCTL polynucleotide and peptide sequence, its fragment, the agonist of the antibody of QPCTL and QPCTL, antagonist or inhibitor can be used as research tool to identify molecular recognition event and the protein of qualification and QPCTL protein-interacting, polypeptide and peptide.Specific examples is phage display peptide library, wherein can filter out in single-wheel time elutriation and be greater than 10 ⁸individual peptide sequence.These class methods known in the art and other method and they can be used for identifying the compound of the activity of the arbitrary QPCTL suppressing or strengthen SEQIDNO:11-18.

The relation of coupling represents functional interaction as mixture or approach, and yeast two-hybrid system can be passed through with the interactional protein of QPCTL, proteomics (otherness 2D gel analysis and mass spectroscopy) and genomics (differential gen expression by microarray or serial analysis of gene expression method SAGE) are identified.

Be accredited as the functional protein that contacts and interaction method with QPCTL and define screening method basis for the inhibitor of these QPCTL-protein interactions, agonist and antagonist and conditioning agent.

As used herein, term " antagonist " refers to such inhibitor molecules, its with QPCTL in conjunction with time reduce the biology of QPCTL or the amount of immunologic competence or duration of effect, such as reduce the enzymic activity of this peptase cyclisation QPCTL substrate N-terminal place Glu residue or Gln residue.Antagonist can comprise protein, nucleic acid, carbohydrate, antibody or other molecule any of reducing QPCTL effect; Such as, they can comprise the small molecules and the organic compound that are combined also inactivation QPCTL by competitive type or non-competitive mechanism.The micromolecular inhibitor of preferred QPCTL.The most preferably competitive micromolecular inhibitor of QPCTL.

The specific examples of QPCTL activity inhibitor describes in example 4.Inhibitor can be such as the inhibitor of QPCTL cyclase activity, or is the inhibitor of QPCTL to the binding activities of protein in addition, and wherein said protein and QPCTL interact.The specific examples of this type of inhibitor such as can comprise and is formulated into anti-QPCTL antibody, peptide, protein fragments or small peptides acyl group proteinase inhibitor in substratum or non-peptide low molecular organic depressant, so that the cell type needed for introducing.Alternatively, this type of inhibitor can be connected with targeting ligand to be imported by cell-mediated endocytosis or other receptor mediated events.These class methods are hereafter further describing, and in view of QPCTL Nucleotide disclosed herein and aminoacid sequence, it can be implemented by those skilled in the art.

Other purposes of QPCTL is the potential antagonist that screening is used as therapeutant, such as, for suppressing with the combination of QPCTL and for screening agonist.QPCTL, its immunogenic fragments or its oligopeptides to may be used in arbitrary multi-medicament triage techniques screening as expecting the library of compound of agonist or antagonist.The fragment used in this type of sieve method can be dissociated in the solution, be fixed to solid support, be carried on cell surface or be positioned at born of the same parents.Measure the formation of associativity mixture between QPCTL and institute's test substances subsequently.Being intended to discovery can suppress other assay method of the antagonist of QPCTL apparent from the disclosure of patent No. WO2004/098625, WO2004/098591 and WO2005/075436, and wherein said patent describes the inhibitor of QC and described patent is intactly incorporated herein.Another important use of these QPCTL is that screening QC inhibitor does not have undesirable side effect to prove them to suppress one or more QPCTL equally.

Be provided for screening the method for molecule that Small molecular libraries identifies in conjunction with QPCTL and generally comprise: a) provide Small molecular libraries; B) by the polypeptide of described Small molecular libraries and SEQIDNO:11-18 or with its fragment carry be suitable for condition that mixture formed under merge; And c) detecting mixture formation, the existence of wherein said mixture determines the small molecules be combined with described QPCTL.

A kind ofly identify that the method for antagonist is included in cracking and send together with chromogenic substrate (such as Ala-Pro-AFC or Ala-Pro-AMC) extract being handed to and coming from cell by the small molecules in conjunction with QPCTL by under the condition normally occurred, wherein said cell is to express the vector of QPCTL, and measure the suppression of the splitting action to described enzyme by the change of monitoring change in fluorescence or UV photoabsorption subsequently, thus identified the molecule suppressing splitting action by spectrophotometry.When there is described molecule, speed of reaction reduces or the total amount reduction of fluorescence or UV photoabsorption then determines that this small molecules is the antagonist reducing QPCTL catalytic activity/enzymic activity, once identify this quasi-molecule, then can use them to reduce or suppress QPCTL to the cyclisation of aminoterminal Glu residue or Gln residue.

According to another concrete aspect, the invention provides the method for SCREENED COMPOUND, described compound can suppress the enzymic activity of at least one maturation protein of the present invention, wherein said maturation protein is preferably selected from SEQIDNO:11-18, described method is included in maturation protein and the suitable substrates for this maturation protein described in incubation when there is one or more test compounds or its salt, measure the enzymic activity of described maturation protein, by this active with lacking the commeasurable expression activitiy that measures under test compounds and selecting to reduce test compounds or the compound of described enzymic activity.

In addition, the present invention also provides the method for screening selectivity QC inhibitor (namely can suppress the compound of QC enzymic activity), wherein said QC is preferably the protein of SQIDNO:10, it does not suppress the enzymic activity of at least one maturation protein of the present invention, wherein said maturation protein is preferably selected from the protein of SEQIDNO:11-18, described method is included in maturation protein and suitable substrates described in incubation when there is one or more inhibitor or its salt, measure the enzymic activity of described maturation protein, by this active with lacking the commeasurable expression activitiy that measures under described QC inhibitor and selecting the compound of the enzymic activity not reducing described maturation protein.

In addition, the present invention also provides the method for the compound of the enzymic activity of screening selectivity QPCTL inhibitor and at least one QPCTL albumen, wherein said QPCTL albumen is preferably the protein of SQIDNO:10, it does not suppress the enzymic activity of QC, wherein said QC is preferably selected from the protein of SEQIDNO:11-18, QC described in incubation when described method is included in one or more inhibitor or its salt of there is QPCTL, measure the enzymic activity of QC, by this active with lacking the commeasurable expression activitiy that measures under described QPCTL inhibitor and selecting the compound of the enzymic activity not reducing described QPCTL albumen.

The useful inhibitor of QC describes in WO2004/098625, WO2004/098591, WO2005/039548 and WO2005/075436, wherein said QC inhibitor also can be used as the inhibitor of QPCTL, described document is intactly incorporated herein, especially with regard to the structure of described inhibitor and generation aspect thereof.

The example of QPCTL-inhibitor

The potential QPCTL-inhibitor of suitable purposes of the present invention and method discloses in WO2005/075436, and described document is intactly incorporated herein with regard to the structure of QC inhibitor, synthesis and application process aspect.

Particularly:

Suitable compound is formula 1 ^*compound:

formula 1 ^*

In still another embodiment, the inhibitor of QPCTL can be those inhibitor of formula 1a,

Wherein R defines in example 1-53.

Example	R	ESI-MS(M+H)
			1	Methyl	199.3
2	The tertiary butyl	241.4
			3	Benzyl	275.4
4	Phenyl	261.4
			5	4-(fluorine)-phenyl	279.35
6	4-(chlorine)-phenyl	295.80
			7	4-(ethyl)-phenyl	289.41
8	4-(trifluoromethyl)-phenyl	329.4
			9	4-(methoxycarbonyl)-phenyl	319.4
10	4-(ethanoyl)-phenyl	303.4
			11	4-(methoxyl group)-phenyl	291.4
12	Dicyclo [2.2.1]-5-in heptan alkene 2-base	277.5
			13	3,4-(dimethoxy)-phenyl	321.5
14	2,4-(dimethoxy)-phenyl	321.5
			15	3,5-(dimethoxy)-phenyl	321.5
16	2-(methoxycarbonyl)-phenyl	319.4
			17	4-(oxazole-5-base)-phenyl	328.5
18	4-(pyrazol-1-yl)-phenyl	327.4
			19	4-(sec.-propyl)-phenyl	303.5
20	4-(piperidines-1-alkylsulfonyl)-phenyl	408.6
			21	4-(morpholine-4-base)-phenyl	346.5
22	4-(cyano group)-phenyl	286.4
			23	2,3-dihydro-benzo [Isosorbide-5-Nitrae] dioxin-6-base	319.4
24	Benzo [1,3] dioxole-5-base	305.4
			25	3,4,5 (trimethoxy)-phenyl	351.5
26	3-(methoxyl group)-phenyl	291.4

Example	R	ESI-MS(M+H)
			27	4-(oxyethyl group)-phenyl	305.5
28	4-(benzyloxy)-phenyl	367.5
			29	4-(methoxyl group)-benzyl	305.5
30	3,4-(dimethoxy)-benzyl	335.5
			31	2-(methoxycarbonyl)-thiene-3-yl-	325.5
32	33-(ethoxy-carbonyl)-4,5,6,7-tetrahydro benzo [b] thiophene-2-base	392.6
			33	2-(methoxycarbonyl)-4-(methyl)-thiene-3-yl-	339.5
34	Benzo [c] [1,2,5] thiazole-4-yl	319.5
			35	Benzo [c] [1,2,5] thiazole-5-base	319.5
36	5-(methyl)-3-(phenyl)-isoxazole-4-base	342.5
			37	3,5-(dimethyl)-isoxazole-4-base	280.4
38	4-(iodine)-phenyl	387.3
			39	4-(bromine)-phenyl	340.3
40	4-(methyl)-phenyl	275.4
			41	Naphthalene-1-base	311.5
42	4-(nitro)-phenyl	306.4
			43	Butyl	241.4
44	Ring octyl group	295.5
			45	Furans-2-ylmethyl	265.4
46	Tetrahydrofuran (THF)-2-ylmethyl	269.4
			47	Benzo [1,3] dioxole-5-ylmethyl	319.4
48	2-(morpholine-4-base)-ethyl	298.5
			49	4-(methyl mercapto)-phenyl	307.5
50	4-(dimethylamino)-phenyl	304.5
			51	4-(trifluoromethoxy)-phenyl	345.4
52	Benzoyl	288.3
			53	Pyrimidine-4-yl	261.1

Other suitable inhibitors of QPCTL can be the inhibitor of formula 1b,

Wherein R ¹and R ²define in example 54-95.

Example	R ¹	R ²
			54	Cyano group	Methyl
55	Cyano group	3,4-(dimethoxy)-phenyl
			56	Cyano group	2,4-(dimethoxy)-phenyl
57	Cyano group	3,5-(dimethoxy)-phenyl
			58	Cyano group	2,3-dihydrobenzo [b] [Isosorbide-5-Nitrae] dioxin-7-base
59	Cyano group	Benzo [d] [1,3] dioxole-6-base
			60	Cyano group	3,4,5-(trimethoxy)-phenyl
61	Cyano group	3-(methoxyl group)-phenyl
			62	Cyano group	4-(oxyethyl group)-phenyl
63	Cyano group	4-(benzyloxy)-phenyl
			64	Cyano group	Phenyl
65	Cyano group	4-(methoxyl group)-phenyl
			66	Cyano group	4-(ethanoyl)-phenyl
67	Cyano group	4-(nitro)-phenyl
			68	Cyano group	Benzyl
69	Cyano group	Naphthalene-1-base
			70	Cyano group	4-(fluorine)-phenyl
71	Cyano group	4-(iodine)-phenyl
			72	Cyano group	4-(bromine)-phenyl
73	Cyano group	Ring octyl group
			74	Cyano group	The tertiary butyl
75	Cyano group	4-(methyl)-phenyl
			76	Cyano group	4-(methylthio group)-phenyl
77	Cyano group	4-(ethyl)-phenyl
			78	Cyano group	4-(dimethylamino)-phenyl

Example	R ¹	R ²
			79	Cyano group	Butyl
80	Cyano group	Trityl
			81	Cyano group	(benzo [d] [1,3] dioxole-6 base) methyl
82	Cyano group	(tetrahydrofuran (THF)-2 base) methyl
			83	Cyano group	4-(trifluoromethyl)-phenyl
84	Cyano group	(furans-2-base) methyl
			85	Cyano group	2-(morpholine-4-base)-ethyl
86	Cyano group	4-(oxazole-5-base)-phenyl
			87	Cyano group	Pyrimidin-3-yl
88	Cyano group	4-(cyano group)-phenyl
			89	Cyano group	4-(trifluoromethoxy)-phenyl
90	Cyano group	4-(piperidines sulphonyl)-phenyl
			91	Cyano group	4-(1H-pyrazol-1-yl) phenyl
92	H	3,4-(dimethoxy)-phenyl
			93	Methyl	3,4-(dimethoxy)-phenyl
94	Cyano group	2,3,4-(trimethoxy)-phenyl
			95	Cyano group	Suberyl

Other suitable inhibitors of QPCTL can be the inhibitor of formula 1c,

Wherein R ³define in example 96-102.

Example	R ³	ESI-MS(M+H)
			96	Ethyl	197.3
97	The fluoro-4H-benzo [d] of 6-[1,3] dioxin-8-bases	321.4
			98	3-(cyclopentyloxy)-4-(methoxyl group)-phenyl	359.4
99	4-(oxygen base in heptan)-phenyl	359.5
			100	3,4-dihydro-2H-benzo [b] [Isosorbide-5-Nitrae] dioxane-7-in heptan base	317.4
101	4-(butoxy)-phenyl	317.4

Example	R ³	ESI-MS(M+H)
			102	3,4-(dimethoxy)-phenyl	305.4

Other suitable inhibitors of QPCTL can be the inhibitor of formula 1d,

Position on wherein said ring defines in 103-105 example.

Example	The position that benzyl replaces	ESI-MS(M+H)
			103	2	383.5
104	3	383.5
			105	4	383.5

Other suitable inhibitors of QPCTL can be the inhibitor of formula 1e,

Wherein R ⁴and R ⁵define in example 106-109.

Other suitable inhibitors of QPCTL can be the inhibitor of formula 1f,

Wherein R ⁶define in example 110-112.

Example	R ⁶	ESI-MS(M+H)
			110	H	259.4
111	Chlorine	293.8
			112	Methoxyl group	289.4

Other suitable inhibitors of QPCTL can be the inhibitor of formula 1g,

Wherein R ⁷, R ⁸and R ⁹define in example 113-132.

Other suitable inhibitors of QPCTL can those inhibitor of formula 1h,

Wherein n defines in example 133-135.

Example	N	ESI-MS(M+H)
			133	3	306.4
134	4	320.5
			135	5	334.5

Other suitable inhibitors of QPCTL can be the inhibitor of formula 1i,

Wherein m defines in 136 and 137 examples.

Example	m	ESI-MS(M+H)
			136	2	307.4
137	4	335.5

Other suitable inhibitors of QPCTL can be the inhibitor of formula 138-141.

As used herein, term " agonist " refers to such inhibitor molecules, its with QPCTL in conjunction with time increase or extend time length of QPCTL effect.Antagonist can comprise and to be combined with QPCTL and to regulate the protein of its performance, nucleic acid, carbohydrate, antibody or other molecule any.Although proof small molecules is that the possibility of QPCTL agonist is less, but to comprise as the method for agonist for the identification of this small molecules in conjunction with QPCTL in the cell that the micromolecular form of adding lustre in conjunction with QPCTL sent and is handed to and transforms with the carrier of expressing QPCTL, and by spectrophotometry fluorescence or UV photoabsorption change.The increasing amount of UV absorption or fluorescence, determines that described small molecules is the agonist improving QPCTL activity.

As described in disclosed PCT application WO84/03564, spendable another kind of drug screening technology provides high flux screening method to such compound, and wherein said compound has suitable binding affinity to target protein matter.In the process, in the different small molecules test compounds that solid substrate (as plastics pin or some other surface) upper synthesis is a large amount of.Described test compounds and QPCTL or its fragment are reacted, and are washed away subsequently.In conjunction with QPCTL detected by method well known in the art subsequently.The QPCTL of purifying also can direct coated at the flat board in foregoing pharmaceutical triage techniques.Alternatively, nonneutralizing antibody can be used to catch peptide and to be fixed on solid support by described peptide.

In another embodiment, competitive drug screening assay assay method can be used, wherein can compete combination to QPCTL in conjunction with the neutralizing antibody of QPCTL and test compounds.By this way, antibody can be used to detect any existence having the peptide of one or more antigenic determinant with QPCTL.

As implied above, by research binding site, can design such part, this part such as has the interaction larger than QPCTL native ligand to QPCTL.This type of antagonist ligand will be combined with QPCTL with higher avidity and thus serve as competitive part.Alternatively, can design and the ligand binding site homology of natural QPCTL or similar synthetic protein or recombinant protein, as other molecule QPCTL to high-affinity can be designed.This quasi-molecule also should be replaced QPCTL and provide protectiveness effect.

As implied above, the structure of understanding QPCTL can the homologue of design and synthesis binding site and analogue.This quasi-molecule will promote to utilize binding characteristic to carry out the potential therapeutant of target greatly, and they also can be used for screening potential therapeutant.In addition, they can be used as the aborning immunogen of monoclonal antibody, and described antibody itself can be used in as in aforesaid diagnosis/herein or therapy.

Therepic use

4 amyloid known in the art such as A β 1-42 (SEQIDNO:23) and A β 1-40 (SEQIDNO:24) with the brachymemma of aminoterminal mode, produce A beta-peptide 3-42 (SEQIDNO:25), 3-40 (SEQIDNO:26), 11-42 (SEQIDNO:27) and 11-40 (SEQIDNO:28) by protein lyase such as aminopeptidase or dipeptides amido peptase.The A β peptide of these brachymemmas start from aminoterminal place glutaminic acid residue and be therefore QC substrate (also see WO2004/09862) and may or SEQIDNO:11-18, the QPCTL of 21 and 22, preferably SEQIDNO:11, the people isoQC of 12,21 and 22, most preferably SEQIDNO:11 and 12 the substrate of people isoQC.The pGlu-A β peptide of gained SEDIDNOS29-32 has much bigger hydrophobicity than the peptide of non-Pyrrolidonecarboxylic acid, very easily in formation A-β peptide aggregation thing, as oligomer and protofibril (fibrill), and proves to have height neurotoxicity.Finally, the A beta-peptide of SEQIDNO:29-32 is played an important role in alzheimer's disease and mongolism development.

Therefore, can by SEQIDNO:11-18, the QPCTL of 21 and 22, preferably SEQIDNO:11,12, the people isoQC of 21 and 22, most preferably SEQIDNO:11 and 12 the inhibitor of people isoQC to be used for the treatment of 4 amyloid diseases related, especially neurodegenerative disease, particularly alzheimer's disease and mongolism.

QPTCL other potential physiological substrate in Mammals is selected from by Glu ¹-ABri (SEQIDNO:33), Glu ¹-ADan (SEQIDNO:34) and Gln ¹the group that-tert-Amyloxycarbonyltetragastrin (17 and 34) (SEQIDNO:35 and 36) forms.Their Pyrrolidonecarboxylic acid form (SEQIDNO:37-40) causes such disease, its be selected from by with or the group that forms without the duodenal cancer of helicobacter pylori infection, colorectal cancer, Zolliger-Ellison syndrome, Familial British Dementia (FBD) and Familial Danish Dementia (FDD) in those diseases.Therefore, the inhibitor of QPCTL can be used to treat these diseases.

Other potential physiological substrate of QPTCL is shown in table 3.

Table 3: the aminoacid sequence of the physiologically active peptide of band aminoterminal glutamine residue

peptide	aminoacid sequence	function
			tert-Amyloxycarbonyltetragastrin 17 (SEQ ID NO:35) Swiss-Prot:P01350	qGPWL EEEEEAYGWM DF (acid amides)	tert-Amyloxycarbonyltetragastrin stimulate stomach mucous membrane to produce and secrete hydrochloric acid and stimulating pancreas secretion digestive ferment.It also stimulates smooth muscle contraction in stomach and intestines and increases blood circulation and moisture is secreted.
neurotensin (SEQ ID NO:41) Swiss-Prot:P30990	qLYENKPRRP YIL	neurotensin plays internal secretion or paracrine action in metabolism of fat regulates.It causes smooth muscle contraction.
			fPP	qEP acid amides	the tripeptides relevant to thyrotrophin-releasing hormone (TRH), is present in refining.Show with external evidence in the body of nearest acquisition that FPP plays a significant role in adjustment sperm fertilizability.
tRH Swiss-Prot:P20396	qHP acid amides	the function of TRH is as the biosynthetic instrumentality of TSH in prepituitary gland and the neurotransmitter/neuromodulator of unifying in peripheral nervous system as central nervous system
			gnRH (SEQ ID NO:42) Swiss-Prot:P01148	qHWSYGL RP (G) acid amides	stimulate gonadotrophin secretion; It stimulates the secretion of prolan B and follicle stimulating hormone.
cCL16 (small molecules can inducing cytokine A16) (SEQ ID NO:43) Swiss-Prot:O15467	qPKVPEW VNTPSTCCLK YYEKVLPRRL VVGYRKALNC HLPAIIFVTK RNREVCTNPN DDWVQEYIKD PNLPLLPTRN LSTVKIITAK NGQPQLLNSQ	display to lymphocyte and monocytic chemotactic activity, but does not show chemotactic activity to neutrophilic granulocyte.Also show strong bone marrow depression active, suppress Meloid progenitor propagation.Restructuring SCYA16 display to monocyte and the monocytic chemotactic activity of THP-1, but does not show chemotactic activity to presence lymphocyte and neutrophilic granulocyte.Eliciting calcium flux in the THP-1 cell desensitized by expressing RANTES in advance.
			cCL8 (small molecules can inducing cytokine A8) (SEQ ID NO:44) Swiss-Prot:P80075	qPDSVSI PITCCFNVIN RKIPIQRLES YTRITNIQCP KEAVIFKTKR GKEVCADPKE RWVRDSMKHL DQIFQNLKP	attract the chemokine of monocyte, lymphocyte, basophilic granulocyte and eosinophilic granulocyte.Can play a role in tumorigenesis and inflammatory host response.This protein can heparin-binding.
cCL2 (small molecules can inducing cytokine A2) (SEQ ID NO:45) Swiss-Prot:P13500	qPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT	attract monocyte and basophilic granulocyte, but do not attract the chemokine of neutrophilic granulocyte or eosinophilic granulocyte.Promote monocyte anti-tumor activity.Be that the disease of feature is (as psoriatic, class with monocyte infiltration

peptide	aminoacid sequence	function
					rheumatic arthritis or atherosclerosis) pathological development in show effect.Convene monocyte in ductus arteriosus wall during can participating in atherosclerotic lysis.Be combined with CCR2 and CCR4.
cCL18 (small molecules can inducing cytokine A18) (SEQ ID NO:46) Swiss-Prot:P55774	qVGTNKELC CLVYTSWQIP QKFIVDYSET SPQCPKPGVI LLTKRGRQIC ADPNKKWVQK YISDLKLNA	attract lymphocyte but do not attract monocyte or granulocytic chemokine.Can participate in B cell folliculus that B cell travels in lymphoglandula.In lymphoglandula, attract ortho states T lymphocyte to dendritic cell and activating macrophage, have the chemotactic activity of naive T cells, CD4+ and CD8+T cell and therefore all can play a role in humoral immunoresponse(HI) and cell-mediated immune response.
			cXXXC chemotactic molecule (neural chemokine) (SEQ ID NO:47) Swiss-Prot:P78423	qHHGVT KCNITCSKMT SKIPVALLIH YQQNQASCGK RAIILETRQH RLFCADPKEQ WVKDAMQHLD RQAAALTRNG GTFEKQIGEV KPRTTPAAGG MDESVVLEPE ATGESSSLEP TPSSQEAQRA LGTSPELPTG VTGSSGTRLP PTPKAQDGGP VGTELFRVPP VSTAATWQSS APHQPGPSLW AEAKTSEAPS TQDPSTQAST ASSPAPEENA PSEGQRVWGQ GQSPRPENSL EREEMGPVPA HTDAFQDWGP GSMAHVSVVP VSSEGTPSRE PVASGSWTPK AEEPIHATMD PQRLGVLITP VPDAQAATRR QAVGLLAFLG LLFCLGVAMF TYQSLQGCPR KMAGEMAEGL RYIPRSCGSN SYVLVPV	soluble form has chemotaxis to T cell and monocyte, but to neutrophilic granulocyte without chemotaxis.Film combining form promotes that these white corpuscles and endotheliocyte adhere to.Can play a role in adjustment endothelium place leukocyte adhesion and process of dividing a word with a hyphen at the end of a line.Be combined with cx3cr1.
cCL7 (small molecules can inducing cytokine A7) (SEQ ID NO:48)	qPVGINT STTCCYRFIN KKIPKQRLES YRRTTSSHCP REAVIFKTKL DKEICADPTQ	attract to drench monocyte and eosinophilic granulocyte, but do not attract the chemokine of neutrophilic granulocyte.Promote monocyte anti-tumor activity.Also induce Gelatinase B.This albumen

peptide	aminoacid sequence	function
			swiss-Prot:P80098	kWVQDFMKHL DKKTQTPKL	matter can heparin-binding.Be combined with CCR1, CCR2 and CCR3.
appetite peptide A (inferior colliculus secretin-1) (SEQ ID NO:49) Swiss-Prot O43612	qPLPDCCRQK TCSCRLYELL HGAGNHAAGI LTL	ingest and the neuropeptide that plays a significant role in sleep-wake regulating, it can play a role by coordinating the complex behavior response of these complementary steady state function and physiologic response.This neuropeptide also plays more wide application in energy metabolism steady-state adjustment, autonomic function, hormonal equilibrium and Humoral immunity.Appetite peptide-A is all combined with OX1R and OX2R with high-affinity.
			arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 (SEQ ID NO:50)	rPK PQQFFGLM	belong to Hormone Peptide.Hormone Peptide is excitor nerve unit, wakes the bioactive peptide of behavior response up, is strong vasodilator and secretogogue and (directly or indirectly) makes several kind of smooth muscle cells shrink.

Peptide Gln ¹-tert-Amyloxycarbonyltetragastrin (17 and 34 amino acid lengths), Gln ¹-neurotensin and Gln ¹-FPP is accredited as the novel physiological substrate of QPCTL.Tert-Amyloxycarbonyltetragastrin, neurotensin and FPP are included in the pGlu residue at N-terminus position place.This aminoterminal pGlu residue can be formed the katalysis of whole above-mentioned peptide from aminoterminal glutamine by QPCTL.Therefore, with regard to its biological function, these peptides activate when described aminoterminal glutamine residue changes into pGlu.

Transepithelial transduction sexual cell, especially tert-Amyloxycarbonyltetragastrin (G) cell, arrive at food under one's belt and coordinate gastric acid secretion.Nearest research confirms that various active product produces from tert-Amyloxycarbonyltetragastrin precursor, and there is multiple reference mark in the biosynthesizing of tert-Amyloxycarbonyltetragastrin.Biosynthesizing precursor and intermediate (tert-Amyloxycarbonyltetragastrin former and Gly-tert-Amyloxycarbonyltetragastrin) are presumption somatomedins; The epithelial propagation of its product-amidation tert-Amyloxycarbonyltetragastrin-regulate, produce acid stomach parietal cell and histamine secretion intestines addicted to the differentiation of chromium sample (ECL) cell and synthesize with histamine in ECL cell and preserve relevant genetic expression, and sharply stimulating acid is secreted.Tert-Amyloxycarbonyltetragastrin also stimulate generation Urogastron (EGF) family member, and the latter transfers to suppress parietal cell function, also stimulate superficial epithelial cells growth.PG concentration raises in the object with helicobacter pylori, and wherein known described object has duodenal ulcer disease and the cancer of the stomach risk (Dockray, G.J.1999JPhysiol15315-324) of increase.

Known peptide hormone-tert-Amyloxycarbonyltetragastrin from G cell in gastric antrum release secrete the synthesis of histamine sour mucous membrane and the release from ECL cell by CCK-2 receptor for stimulating.Mobilize histamine by with H (2) receptors bind on parietal cell and induce acid secrete.Recent research shows that the complete amidated form of tert-Amyloxycarbonyltetragastrin or less form processing (the former tert-Amyloxycarbonyltetragastrin extended with glycine of tert-Amyloxycarbonyltetragastrin) are also GI somatomedins.The Major Nutrient effect of having established amidation tert-Amyloxycarbonyltetragastrin secretes sour mucous membrane for stomach, causes stomach stem cell and ECL cell proliferation to increase at described amidation tert-Amyloxycarbonyltetragastrin in sour mucous membrane of secreting, and causes the stomach hole cell that increases and ECL cell quality.In other words, the Major Nutrient target of processing less tert-Amyloxycarbonyltetragastrin (tert-Amyloxycarbonyltetragastrin that such as glycine extends) seemingly mucous membrane of colon (Koh, T.J. and Chen, D.2000RegulPept9337-44).

In still another embodiment, the invention provides the purposes increasing active QPCTL effector, for the cell proliferation of stimulating gastrointestinal road, especially produce acid stomach parietal cell propagation and histamine secretion intestines addicted to chromium sample (ECL) cell proliferation, produce acid stomach parietal cell and secretion histamine intestines addicted to the differentiation of chromium sample (ECL) cell and synthesize to histamine in ECL cell and preserve relevant gene expression and for by maintaining or increasing active pGlu ¹the concentration of-tert-Amyloxycarbonyltetragastrin (SEQIDNO:39 and 40) and sharply stimulating acid secretion in Mammals.

In still another embodiment, the invention provides the purposes of the inhibitor of QPCTL, reducing non-activity Gln for passing through ¹-tert-Amyloxycarbonyltetragastrin (SEQIDNO:35 and 36) are to active pGlu ¹the conversion of-tert-Amyloxycarbonyltetragastrin (SEQIDNO:39 and 40) and treat in Mammals with or without the duodenal ulcer disease of helicobacter pylori infection and cancer of the stomach.

Neurotensin (NT) (SEQIDNO:41) relates to the neuropeptide of the specificity adjustment neurotransmitter system of schizophrenia physiopathology, and wherein said neurotransmitter system had previously confirmed functional disorder in described illness.The clinical study of having measured cerebrospinal fluid (CSF) NT concentration discloses schizophreniac's subgroup of the CSFNT concentration with reduction, and wherein said CSFNT concentration is recovered by effective antipsychotics therapy.Also exist and participate in the consistent a large amount of evidences of the antipsychotics mechanism of action with NT system.The NT behavior effect that maincenter mode is used is obviously similar to the antipsychotics of systemic administration with biological chemistry effect, and antipsychotics increases NT neurotransmission.The association of this result of study produces such hypothesis: NT plays a role as endogenous anti-semen antibody.In addition, typical case and atypical antipsychotic otherness ground change the NT neurotransmission in nigrostriatum and middle limbic brain Dopamine HCL terminal zone, and these effects predict side effect susceptibility and curative effect (2001BiolPsychiatry50856-872 such as Binder, E.B.) respectively.

Therefore, the invention provides the purposes increasing active QPCTL effector, for the preparation of antipsychotics and/or for treating schizophrenia in Mammals.Described QPCTL effector maintains or increases active pGlu ¹the concentration of-neurotensin.

The tripeptides relevant to thyrotrophin-releasing hormone (TRH) is present in refining.Show with external evidence in nearest acquisition body that FPP plays a significant role in adjustment sperm fertilizability.Specifically, FPP initially stimulates without fertilization property (non-capacitation) sperm " transformation " and more promptly becomes and can educate, but causes capacitation stagnation subsequently, thus sperm spontaneous acrosome loss does not occur and thus do not lose fertility.These responses are simulated by adenosine and are in fact strengthened by it, and wherein known adenosine regulates adenylyl cyclase (AC)/cAMP signal transduction pathway.Proved that FPP and adenosine all stimulate the generation of cAMP in non-capacitation sexual cell, but in capacitation cell, then suppressed it to produce, FPP acceptor interacts to realize the adjustment to AC with Adenosine Receptors and G-protein to a certain degree simultaneously.The tyrosine phosphorylation state of these events affecting multiple proteins, wherein some protein is important in initial " transformation ", and other oroteins may participate in acrosomal reaction itself.Also be present in thyrocalcitonin in refining and Angiotensin II to non-capacitation sperm, there is similar in vitro effects and the response to FPP can be promoted.These molecules have effect in similar body, by stimulating and maintaining fertility subsequently and affect fertilizability.The availability of FPP, adenosine, thyrocalcitonin and Angiotensin II reduces or its acceptor defect causes male sterile (Fraser, L.R. and Adeoya-Osiguwa, S.A.2001VitamHorm63,1-28).

In still another embodiment, the inhibitor that the invention provides QPCTL suppresses medicine for the preparation of fertilization and/or in Mammals, reduces the purposes of fertilizability.Described QPCTL inhibitor reduces active pGlu ¹the concentration of-FPP, thus cause preventing capacitation and inactivation spermoblast.On the contrary, what can confirm is increase active QC effector and can to treat sterile in male moderate stimulation fertilizability.

In still another embodiment, other physiological substrate of QPCTL is identified in the present invention.These physiological substrate are Gln ¹-CCL2 (SEQIDNO:45), Gln ¹-CCL7 (SEQIDNO:48), Gln ¹-CCL8 (SEQIDNO:44), Gln ¹-CCL16 (SEQIDNO:43), Gln ¹-CCL18 (SEQIDNO:46) and Gln ¹-CXXXC chemotactic molecule (SEQIDNO:47).Refer to table 2.Suppression, tumorigenesis, inflammatory host response, cancer, psoriatic, rheumatoid arthritis, atherosclerosis, humoral immunoresponse(HI) and cell-mediated immunne response, leukocyte adhesion that these polypeptide are bred as Meloid progenitor at pathophysiological condition and play a significant role in the process of dividing a word with a hyphen at the end of a line at endothelium place.

Have studied several for hepatitis B virus, human immunodeficiency virus and the melanomatous vaccine based on cytotoxic T cell peptide recently in clinical trial.Separately or be decapeptide ELA with a kind of interesting melanoma candidate vaccine that other tumour antigen combines.This peptide is Melan-A/MART-1 antigen immune advantage peptide analogs, band aminoterminal L-glutamic acid.Report that the amino of L-glutamic acid and the amino of γ-carboxyl and glutamine and the easy condensation of γ-formamido-are to form pyroglutamic acid derivatives.For solving this stability problem, developed several pharmacy object peptide, it has the Pyrrolidonecarboxylic acid of alternative aminoterminal L-glutamic acid or glutamine, and does not lose pharmacological characteristics.But compared with ELA, described pyroglutamic acid derivatives (PyrELA) and in addition aminoterminal ethanoyl add cap derivative (AcELA) can not activated cell poison T lymphocyte (CTL) activity.Even if imported apparent trickle modification in PyrELA and AcELA, but these two kinds of derivatives may have comparatively low-affinity to specific MHC I type than ELA.Therefore, in order to retain whole activity of ELA, PyrELA (BeckA. etc. 2001, JPeptRes57 (6): 528-38.) must be avoided the formation of.Recently, enzyme glutaminyl cyclase (QC) also overexpression (RossD.T etc., 2000, NatGenet24:227-35.) in melanoma is found.

Therefore, the invention provides the purposes of inhibitor for the preparation of medicine of QPCTL, described medicine is used for the treatment of pathophysiological condition, as the suppression of Meloid progenitor propagation, tumorigenesis, inflammatory host response, cancer, pernicious transfer, melanoma, psoriatic, rheumatoid arthritis, atherosclerosis, impaired humoral immunoresponse(HI) and cell-mediated immunne response, leukocyte adhesion and dividing a word with a hyphen at the end of a line at endothelium place.

In addition, Gln ¹-appetite peptide A (SEQIDNO:49) is accredited as the physiological substrate of the QPCTL in the scope of the invention.Appetite peptide A ingests and the neuropeptide that plays a significant role in sleep-wake regulating, and it can play a role by coordinating the complex behavior response of these complementary steady state function and physiologic response.This neuropeptide also plays a role in energy metabolism steady-state adjustment, autonomic function, hormonal equilibrium and Humoral immunity.

In still another embodiment, the invention provides the purposes of inhibitor for the preparation of medicine of QPCTL, described medicine be used for the treatment of ingest and sleep-wake impaired, energy metabolism steady-state adjustment is impaired, autonomic function is impaired, hormonal equilibrium is impaired and Humoral immunity effect is impaired.

Polyglutamyl amine extension in some protein causes neurodegenerative disease, as Parkinson's disease and Kennedy disease.Its mechanism is largely still unknown.The biochemical characteristic of polyglutamyl amine tumor-necrosis factor glycoproteins proposes a kind of possible explanation: decompose cracking at glutamyl-glutamy base key in place, form pyroglutamate subsequently, this can contribute to pathological development (Saido, T by the metabolic stability of enhancement polyglutamyl base protein, hydrophobicity, amyloidogenic and neurotoxicity; MedHypotheses (2000) Mar; 54 (3): 427-9).Therefore, thus the present invention provides the inhibitor of QPCTL for the preparation of the purposes of medicine for the treatment of Parkinson's disease and Huntington Chorea.

Another substrate of QPTCL is peptide QYNAD (SEQIDNO:51).Its Pyrrolidonecarboxylic acid form pGlu-Tyr-Asn-Ala-Asp (pEYNAD) (SEQIDNO:52) is the active drug of the activity with blocking voltage gated sodium channel.Sodium channel plays special effect with High Cell Density And High Expression in medullated aixs cylinder and in mammal brain and spinal cord in axonal conduction action potential.Therefore, expect that sodium channel participates in some aspects of the physiopathology of multiple sclerosis (MS), Guillain-Barr é syndrome and chronic inflammatory demyelinating polyneuropathy.

In still another embodiment, the invention provides the purposes of inhibitor for the preparation of medicine of QPCTL, described medicine is used for the treatment of Inflammatory Autoimmune disease, be particularly useful for treating multiple sclerosis, Guillain-Barr é syndrome and chronic inflammatory demyelinating polyneuropathy, wherein the formation of the peptide pEYNAD of blocking voltage gated sodium channel is suppressed.

In addition, the invention provides diagnostic assay method, it comprises QC inhibitor.

In another embodiment, the invention provides the method for the arbitrary aforementioned diseases of diagnosis and/or the patient's condition, comprise step

-collect from the doubtful sample suffering from the object of described disease and/or the patient's condition.

-this sample is contacted with QC inhibitor, and

-determine whether this object suffers from described disease and/or the patient's condition.

Preferably, the sample in described diagnostic method is blood sample, serum sample, spinal fluid samples or urine sample.

Preferably, in described diagnostic method to as if people.

Preferably, the QC inhibitor in described diagnostic method is selectivity QC inhibitor.

Also be preferred for being selectivity QPCTL inhibitor in described diagnostic assay method.

The invention still further relates to the diagnostic kit for implementing diagnostic method, wherein said diagnostic method comprises preceding diagnosis assay method as detection means and defining method.

Embodiment 1: the preparation of people isoQC

clone and substratum

African green monkey kidney cell line COS-7, people's neuroblastoma cell line SH-SY5Y, people's astrocytoma cell lines LN405, Human keratinocytes oncocyte system HaCaT and human hepatocellular carcinoma cell line Hep-G2 are at suitable cell culture medium (DMEM, 10%FBS be used for COS-7, SH-SY5Y, LN405, HaCaT), (RPMI1640,10%FBS is used for Hep-G2) in, at 5%CO ₂(HaCaT, Hep-G2, COS-7) or 10%CO ₂37 DEG C of cultivations under the humidified atmosphere of (SH-SY5Y, LN405).

rT-PCR analyst isoQC is used to express

Use the mini test kit of RNeasy (Qiagen) from SH-SY5Y, LN405, HaCaT and Hep-G2 cellular segregation total serum IgE and passed through SuperScriptII (Invitrogen) and carry out reverse transcription.Subsequently, with HerculaseEnhancedDNA-polysaccharase (Stratagene), primer isoQCh-1 is used (to have justice, SEQIDNO:53) and isoQCh-2 (antisense, SEQIDNO:54) in 25 μ l react to 1: 12.5 dilution amplification people isoQC of the cDNA product produced.Utilize the PCR primer of StrataprepPCR Purification Kit Hep-G2 and confirmed by order-checking.

result

RT-PCR analyst isoQC is used to express

The transcript of finder isoQC is present in clone SH-SY5Y (Fig. 6, swimming lane 1), LN405 (Fig. 6, swimming lane 2), HaCaT (Fig. 6, swimming lane 3) and Hep-G2 (Fig. 6, swimming lane 4).The PCR primer of Hep-G2 is confirmed by order-checking.

the separation of people isoQC

Use RT-PCR from Hep-G2 cellular segregation people isoQC full-length cDNA.In brief, the total serum IgE of Hep-G2 cell is by SuperScriptII (Invitrogen) reverse transcription.Subsequently, with HerculaseEnhancedDNA-polysaccharase (Stratagene), primer isoQChu-1 is used (to have justice, SEQIDNO:55) and isoQChu-2 (antisense, SEQIDNO:56) in 25 μ l react to 1: 12.5 dilution amplification people isoQC of the cDNA product produced.By gained PCR-product subclone to carrier pPCRScriptCAMSK (+) (Stratagene) and by order-checking confirmation.

Embodiment 2: the preparation of isoQC and expression in mammaliancellculture

the molecular cloning of the plasmid vector of encoding human isoQC-EGFP fusion rotein

Whole cloning process uses standard molecular biological technique to carry out.For at people's cells people isoQC-EGFP fusion rotein, use carrier pEGFP-N3 (Invitrogen).Merge with the plasmid of coding enhanced green fluorescence protein (EGFP) at methionine(Met) I or at the cDNA of the ortho states people isoQC of methionine(Met) II place beginning in the aminoterminal mode meeting open reading-frame (ORF).Primer isoQCEGFP-1MetI (SEQIDNO:57) and isoQCEGFP-3 (SEQIDNO:59) starts from the people isoQC of methionine(Met) I for increasing and primer isoQCEGFP-2MetII (SEQIDNO:58) and isoQCEGFP-3 (SEQIDNO:59) starts from the people isoQC of methionine(Met) II for increasing.Utilize restriction site EcoRI and SalI, fragment insertion vector pEGFP-N3 (Invitrogen) correct be inserted through order-checking and confirm.Subsequently, EndoFreeMaxi test kit (Qiagen) carrier of separating is used to be used for cell cultures object.

the cloning process of the amino terminal sequence of hisoQC

In addition, utilize EGFP-1 (having justice) (SEQIDNO:85) and EGFP-2 (antisense) (SEQIDNO:86) for increasing, the EGFP sequence of carrier pEGFP-N3 (Invitrogen) is imported carrier pcDNA3.1 (Invitrogen).Described fragment imports the XhoI site of pcDNA3.1.IsoQCEGFP-1MetI is used (to have justice, SEQIDNO:57) and hisoQCSSEGFPpcDNA (antisense) (SEQIDNO:87) as start from methionine(Met) I hisoQC n terminal fragment primer and use isoQCEGFP-2MetII (to have justice, SEQIDNO:58) and hisoQCSSEGFPpcDNA (antisense) (SEQIDNO:87) as the hisoQC n terminal fragment primer starting from methionine(Met) II, the hisoQC amino terminal sequence of each comfortable 53rd the Serine place ending starting from methionine(Met) I and II is merged with the EGFP in carboxyl terminal mode and carrier pcDNA3.1.EcoRI and the NotI restriction site of described fragment insertion vector pcDNA3.1.Subsequently, EndoFreeMaxi test kit (Qiagen) carrier of separating is used to be used for cell cultures object.

for the cloning process of natural expression hisoQC and hQC

In use primer hQC-1 (having justice) (SEQIDNO:82) and hQC-2 (antisense) (SEQIDNO:83) for hQC, primer isoQCEGFP-1MetI is used (to have justice, SEQIDNO:57) and hisoQCpcDNA (antisense) (SEQIDNO:84) for the hisoQC starting from methionine(Met) I, and after using primer isoQCEGFP-2MetII (having justice) (SEQIDNO:58) and hisoQCpcDNA (antisense) (SEQIDNO:84) to increase for the hisoQC starting from methionine(Met) II, by HindIII and the NotI restriction site of natural hQC insertion vector pcDNA3.1 (+) (Invitrogen) and by EcoRI and the NotI restriction site of natural hisoQC insertion vector pcDNA3.1 (+) (Invitrogen).

for the cloning process of hisoQC and hQC of FLAG labeling

People QC with carboxyl terminal FLAG label to increase HindIII and the NotI restriction site of rear clone to carrier pcDNA3.1 in use primer hQC-1 (having justice) (SEQIDNO:82) and hQCC-FLAGpcDNA (antisense) (SEQIDNO:88).People isoQC with carboxyl terminal FLAG label is at use isoQCEGFP-1MetI, (having justice), and hisoQCC-FLAGpcDNA (SEQIDNO:57), (antisense), (SEQIDNO:89) as start from methionine(Met) 1 hisoQC primer and use isoQCEGFP-2MetII, (having justice), and hisoQCC-FLAGpcDNA (SEQIDNO:58), (antisense), (SEQIDNO:89) as start from methionine(Met) 2 hisoQC primer amplification after insertion vector pcDNA3.1.

Embodiment 3: to the immunohistochemical staining of people isoQC in mammalian cell

the transfection of COS-7 and LN405 and histochemical stain

For expressing the people isoQC-EGFP fusion rotein starting from methionine(Met) I or methionine(Met) II, COS-7 and LN405 is cultivated in containing 6 hole culture dish of cover glass.Cell culture to 80% is converged, use Lipofectamin2000 (Invitrogen) to carry out transfection according to manufacturers's handbook and in Transfection solution incubation 5 hours.After this, described solution is replaced with suitable growth medium and overnight culture cell.

Next day, cell D-PBS (Invitrogen) washes twice and uses ice-cold methyl alcohol to fix 10 minutes at-20 DEG C, uses D-PBS to carry out 3 washing steps in room temperature subsequently and continues 10 minutes.For making golgi zone dye, by COS-7 and LN405 and rabbit anti-mannosidase II polyclonal antibody (Chemicon) with 1: 50 antibody dilution incubation 3 hours in D-PBS.For making the plastosome in COS-7 and LN405 dye, by cell and mouse anti human plastosome monoclonal antibody (Chemicon) with 1: 100 antibody dilution incubation at room temperature 3 hours in D-PBS.Subsequently, cell D-PBS washs three times lasting 10 minutes.For the cell of golgi zone dyeing with the goat anti-rabbit igg second antibody of puting together in rhodamine-RedX (Dianova) at room temperature incubation 45 minutes in dark.For the cell of plastosome dyeing with the goat anti-mouse IgG second antibody of puting together in rhodamine-RedX (Dianova) at room temperature incubation 45 minutes in dark.After this, cell D-PBS continues 5 minutes three times in room temperature washing, and cell D-PBS continues 5 minutes for three times in room temperature washing and cover glass is at least encapsulated (mounted) on microslide with Citiflour.Observation of cell under fluorescent microscope (Carl-Zeiss).

result

1.LN405 transfection and histochemical stain

The expression of the people isoQC-EGFP fusion rotein in clone LN405 (green fluorescence) starting from methionine(Met) I and methionine(Met) II causes the compartmentation of gained protein.Use mannosidase II antibody redying (red fluorescence) and superposing people isoQC-EGFP with mannosidase II subsequently the golgi zone of LN405, show that people isoQC-EGFP fusion rotein is positioned in Golgi compartments and (merge the yellow color of image) (Fig. 7,9).Therefore, it is evident that the people isoQC starting from methionine(Met) II place is enough to cause the Golgi localization of people isoQC fusion rotein.

Owing to lacking the yellow color merging image after superposition, therefore start from the people isoQC-EGFP expressing fusion protein (green fluorescence) of methionine(Met) I and II and plastosome and redye (red fluorescence) and there is no to disclose the people isoQC-EGFP fusion rotein starting from methionine(Met) I or II and be positioned at (Fig. 8,10) in plastosome.

The transfection of 2.COS-7 and histochemical stain

Similar with the expression of people isoQC-EGFP fusion rotein in clone LN405 starting from methionine(Met) I and methionine(Met) II, the expression of people isoQC-EGFP fusion rotein in COS-7 starting from methionine(Met) I and methionine(Met) II causes the compartmentation (green fluorescence) of gained protein.The expression (green fluorescence) of people isoQC-EGFP fusion rotein in clone LN405 (green fluorescence) starting from methionine(Met) I and methionine(Met) II causes the compartmentation of gained protein.Use mannosidase II antibody redying (red fluorescence) and superposing people isoQC-EGFP with mannosidase II subsequently the golgi zone of COS-7 cell, show that people isoQC-EGFP fusion rotein is positioned in the Golgi compartments of COS-7 and (merge the yellow color of image) (Figure 11,13).Equally, the people isoQC-EGFP starting from methionine(Met) II place is enough to cause Golgi localization.

As expected, owing to lacking the yellow color merging image after superposition, therefore start from the expression (green fluorescence) of the people isoQC-EGFP fusion rotein of methionine(Met) I and II and plastosome in COS-7 and redye (red fluorescence) and do not cause the people isoQC-EGFP fusion rotein starting from methionine(Met) I or II to be positioned at (Figure 12,14) in plastosome.

Embodiment 4: the expression and purification of people isoQC in intestinal bacteria

host strain and substratum

Use coli strain DH5 α with breed plasmid and coli strain BL21 for expressing people isoQC.Coli strain is cultivated according to manufacturer specification (Qiagen (DH5 α) Stratagene (BL21)), transformed and analyze.Recommend according to manufacturers, prepare the substratum required for intestinal bacteria and Luria-Bertani (LB) substratum.

the molecular cloning of the plasmid vector of encoding human QC

Whole cloning process uses standard molecular biological technique to carry out.For expressing in e. coli bl21, use carrier pET41a (Novagen).The cDNA starting from the one-tenth acquaintance isoQC of the 30th bit codon (counting from methionine(Met) II) merges with the plasmid of coding GST label to meet open reading-frame (ORF) mode.After utilizing primer hisoQCpET41a-1 (SEQIDNO:60) and hisoQCpET41a-2 (SEQIDNO:61) (table 4) amplification, import amino-terminal protein protease cleavage site and the carboxyl terminal (His) of enteropeptidase ₆label.After subclone, utilize restriction site SpeI and EcoRI that described fragment is inserted described expression vector.

expression and purification in e. coli bl21

The construct of encoding human isoQC is converted into BL21 cell (Stratagene) and cultivates on selectivity LB agar plate at 37 DEG C.In containing the LB substratum of 1% glucose, protein expression is implemented at 37 DEG C.Reaching OD ₆₀₀after about 0.8, express 4 hours with 20 μMs of IPTG at 37 DEG C of induction isoQC.Cell is separated from substratum by centrifugal (4000 × g, 20 minutes), is resuspended in PBS (140mMNaCl, 2.7mMKCl, 10mMNa ₂hPO ₄, 1.8mMKH ₂pO ₄, pH7.3) in and carry out cracking by a FrenchPress subsequently by a freeze-thaw cycle.This cell lysate uses phosphatic damping fluid (50mMNa ₂hPO ₄, 500mMNaCl, pH7.3) and be diluted to final volume 1.5L and at 4 DEG C at 13,400 × g upper centrifugal 1 hour.After centrifugal, use Bradford method to measure the protein concn of gained supernatant liquor, as required, described solution is diluted the final total protein concentration obtaining 0.6mg/ml again.Utilize 4-step scheme purifying GST-isoQC fusion rotein (table 5).Purity analyzes display by the SDS-PAGE in Figure 20.

Embodiment 5: for the assay method of glutaminyl cyclase activity

fluorometry

Whole measurement is carried out at 30 DEG C with Microplate NOVOStar readout instrument (BMGLabtechnologies).H-Gln-β NA is used to evaluate QC activity in fluorescence measurement mode.Sample is made up of the QC aliquots containig of 0.25U pyroglutamyl base aminopeptidase (Qiagen, Hilden, Germany) in the raw fluorogenic substrate of the 0.2mM in final volume 250 μ l, 0.05MTris/HCl (pH8.0) and appropriateness dilution.Excitation/emission wavelength is 320/410nm.Assaying reaction starts by adding glutaminyl cyclase.QC activity measures under condition determination from the typical curve of beta-naphthylamine.A unit definition is the amount of the QC that the 1 μm of olpGlu-β NA of per minute catalysis is under the described conditions formed from H-Gln-β NA.

In the second fluorometry, H-Gln-AMC is used to measure QC as substrate active.Microplate NOVOStar readout instrument (BMGLabtechnologies) is utilized to implement reaction at 30 DEG C.Sample is made up of the QC aliquots containig of 0.1U pyroglutamyl base aminopeptidase (Qiagen) in the raw fluorogenic substrate of multiple concentration in final volume 250 μ l, 0.05MTris/HCl (pH8.0) and appropriateness dilution.Excitation/emission wavelength is 380/460nm.Assaying reaction starts by adding glutaminyl cyclase.QC activity measures under condition determination from the typical curve of 7-amino-4-methylcoumarin.Use GraFit software evaluation dynamics data.

the spectrophotometer measurement assay method of isoQC

Use this assay method to measure the kinetic parameter of most QC substrate.Use continous way method (Schilling, S. etc., 2003BiolChem384,1583-1592), utilize glutamate dehydrogenase as auxiliary enzymes, analyze QC activity in spectrophotometry mode.Sample is made up of corresponding QC substrate, 0.3mMNADH, 14mM α-ketoglutaric acid and the 30U/ml glutamate dehydrogenase in final volume 250 μ l.Reaction starts by adding QC and carries out 8-15 minute by monitoring in the decline of 340nm place absorbancy.Under condition determination, evaluate initial velocity and measure enzymic activity from the typical curve of ammonia.Microplate Sunrise readout instrument is used to measure whole sample at 30 DEG C.Use GraFit software evaluation dynamics data.

inhibitor assay method

For inhibitor inspection, sample composition is same as above, except adding the inhibition compound of presumption.For quick test QC restraining effect, sample contains various inhibitor and the 1K of 4mM _mconcentration of substrate.For studying described restraining effect in great detail and measuring K _i-value, first studies the impact of described inhibitor on described auxiliary enzymes.In each case, on the two kinds of enzymes detected all without impact, therefore, it is possible to reliably determine QC restraining effect.Use GraFit software, by one group of progress curve matching is evaluated suppression constant to the universal equation of competitive inhibition.

result

Multiple different substrate (table 3) is have rated by the transformation of people isoQC.The substrate all analyzed all is transformed by isoQC, shows the overall specificity (Schilling, S. etc., 2003BiolChem384,1583-1592) of the relative loose similar to people QC.As previously to (Schilling observed by people QC; S. etc.; 2003BiolChem384,1583-1592), most high specific constant (k is observed to the substrate (such as Gln-AMC) carrying the large volume hydrophobic amino acid adjoined with aminoterminal glutamyl residue _cat/ K _m).On the contrary, residue electronegative in same position causes specificity significantly to reduce, observed by Gln-Glu, thus reactive site electronegative in instruction isoQC.Compared with people QC, the enzymic activity (Figure 21) that two kinds of restructuring isoQC display is lower.Difference is at the most to an order of magnitude.According to the specificity of isoQC, so hypothesis is rational: this enzyme is responsible for transforming different substrate in body, and namely isoQC participates in the generation of numerous different physiological substrate.

People isoQC activity is by imdazole derivatives competitive inhibition (table 6, Figure 15).The suppression constant K of imidazoles and benzoglyoxaline _iwith the previous value fairly similar obtained people QC.But, K is observed to potent QC inhibitor P150/03 _idecline 10 times.Therefore, the combination model of chelating moiety and imidazole ring seems very similar.Hypothetically, this kind of situation is caused by the complexing of imidazoles basic nitrogen because of the reactive site zine ion of QC and isoQC.The K of P150/03 _ivalue difference is different clearly illustrates that the reactive site of these two kinds of enzymes shows imperceptible difference.Therefore, can produce optionally inhibitor is demonstrated to a kind of enzyme isoform.Selective depressant is of value to the described disease for the treatment of.

Table 3: kinetic assay is to the peptide substrates of people QC and people isoQC.People isoQC expresses in e. coli bl21 (hisoQCdt) or pichia pastoris phaff (YSShisoQC).Substrate shows with amino acid single character code.

Substrate

K _M(mM) hisoQCdt

K _M(mM) YSShisoQC

k _cat(s ^-1) hisoQCdt

k _cat(s ^-1) YSShisoQC

k _cat/K _M(mM ^-1*s ^-1) hisoQCdt

k _cat/K _M(mM ^-1*s ^-1) YSShisoQC

Q-βNA

0,03±0,002

0,035±0,0005

3,37±0,12

8,16±0,87

93,26±6,68

228,70±22,22

QAMC

0,01±0,0009

0,03±0,0064

1,07±0,03

3,72±0,44

62,57±5,68

102,87±29,22

QQ

0,11±0,027

0,11±0,007

2,72±0,25

6,08±0,17

24,50±4,009

54,32±4,61

QE

0,7±0,13

0,61±0,064

2,64±0,21

5,33±0,43

3,85±0,56

8,75±0,87

QG

0,42±0,04

0,36±0,047

1,65±0,04

3,24±0,18

3,93±0,31

9,01±1,75

QGP

0,21±0,016

0,23±0,02

4,01±0,14

8,98±0,07

18,82±1,26

38,42±3,55

QYA

0,22±0,01

0,08±0,022

7,7±0,4

16,47±0,72

66,48±13,07

206,9±57,54

QFA

0,11±0,016

0,104±0,025

7,49±0,28

11,68±2,39

33,03±2,38

116,99±34,37

QEYF

0,03±0,004

0,04±0,004

3,34±0,15

5,64±0,39

109,57±21,03

122,56±5,6

QEDL

0,63±0,052

0,16±0,01

6,41±0,15

9,24±0,65

10,2±0,84

55,04±5,14

Table 4: the primer

Primer	Sequence 5 ' → 3 '	Application
			IsoQCh-1 (SEQ ID NO：53)	GGTCTACACCATTTGGAGCGGCTGGC	Cell line selection
IsoQCh-2 (SEQ ID NO：54)	GGGTTGGAAGTACATCACTTCCTGGGG	Cell line selection
			IsoQChu-1 (SEQ ID NO：55)	ACCATGCGTTCCGGGGGCCGCGGG	The separation of hisoQC
IsoQChu-2 (SEQ ID NO：56)	ACGCTAGAGCCCCAGGTATTCAGCCAG	The separation of hisoQC
			IsoQC EGFP-1 Met I (SEQ ID NO：57)	ATATATGAATTCATGCGTTCCGGGGGCCGC	People isoQC (Met I) is cloned into carrier pEGFP-N3
IsoQC EGFP-2 Met II (SEQ ID NO：58)	ATATATGAATTCATGGAGCCACTCTTGCCGCCG	People isoQC (Met II) is cloned into carrier pEGFP-N3
			IsoQC EGFP-3 (SEQ ID NO：59)	ATATATGTCGACGAGCCCCAGGTATTCAGCCAG	People isoQC (Met I and Met II) is cloned into carrier pEGFP-N3
HisoQC pET41a-1 (SEQ ID NO：60)	ATATACTAGTGATGACGAC GACAAGTTCTACACCATTTGGAGCG	People isoQC is cloned into carrier pET41a
			HisoQC pET41a-2 (SEQ ID NO：61)	TATAGAATTCCTAGTGATGGT GATGGTGATGGAGCCCCAGGTATTCAGC	People isoQC is cloned into carrier pET41a
hisoQC HIS C-Term pPICZAA-1 (SEQ ID NO：62)	ATA TGA ATT CTT CTA CAC CAT TTG GAG C	People isoQC is cloned into carrier PPICZ α A
			hisoQC HIS N-Term pPICZAA-1 (SEQ ID NO：63)	ATA TGA ATT CCA TCA CCA TCA CCA TCA CTT CTA CAC CAT TTG GAG CGG C	People isoQC is cloned into carrier PPICZ α A
hisoQC HIS N-Term pPICZAA-2 (SEQ ID NO：64)	5’-ATA TAT GCG GCC GCC TAG AGC CCC AGG TAT TCA GC-3’	People isoQC is cloned into carrier PPICZ α A
			isoQCm RTs (SEQ ID NO：65)	CCA GGA TCC AGG CTA TTG AG	The real-time PCR analysis of isoQC
hisoQC HIS C-Term pPICZAA-2	ATA TAT GCG GCC GCC TAG TGA TGG TGA TGG TGA TGG AGC CCC AGG TAT TCA GCC AG	People isoQC is cloned into carrier PPICZ α A

Primer	Sequence 5 ' → 3 '	Application
			(SEQ ID NO：66)
isoQCm RTas (SEQ ID NO：67)	TTC CAC AGG GCC GGG GGG C	The real-time PCR analysis of isoQC
			isoQCm MetI s (SEQ ID NO：68)	ATG AGT CCC GGG AGC CGC	The clone of mouse isoQC cDNA
isoQCm MetI as (SEQ ID NO：69)	CTA GAG TCC CAG GTA CTC	The clone of mouse isoQC cDNA
			isoQCm kurz s (SEQ ID NO：70)	AGT TCC TGC CCC TGC TGC TG	The clone of mouse isoQC cDNA
mQC RT s (SEQ ID NO：71)	ATC AAG AGG CAC CAA CCA AC	The real-time PCR analysis of mQC
			mQC RTas (SEQ ID NO：72)	CTG GAT AAT ATT TCC ATA G	The real-time PCR analysis of mQC
MQC RT aminoterminal s (SEQ ID NO:73)	ACA GCT GGG AAT CTG AGT C	The real-time PCR analysis of mQC
			MQC RT aminoterminal as (SEQ ID NO:74)	GAG CAG AAT AGC TTC CGG GCG	The real-time PCR analysis of mQC
Iso-I55Ns (SEQ ID NO：75)	CTG CGG GTC CCA TTG AAC GGA AGC CTC CCC GAA	Site-directed mutagenesis hisoQC I55N
			Iso-I55Nas (SEQ ID NO：76)	TTC GGG GAG GCT TCC GTT CAA TGG GAC CCG CAG	Site-directed mutagenesis hisoQC I55N
Iso-C351As (SEQ ID NO：77)	ACG GTA CAC AAC TTG GCC CGC ATT CTC GCT GTG	Site-directed mutagenesis hisoQC C351A
			Iso-C351Aas (SEQ ID NO：78)	CAC AGC GAG AAT GCG GGC CAA GTT GTG TAC CGT	Site-directed mutagenesis hisoQC C351A
hQC-1 (SEQ ID NO：82)	ATATATAAGCTTATGGCAGGCGGAAGACAC	Natural hQC is inserted pcDNA 3.1
			hQC-2 (SEQ ID NO：83)	ATATGCGGCCGCTTACAAATGAAGATATTCC	Natural hQC is inserted pcDNA 3.1
hisoQC pcDNA as (SEQ ID NO：84)	ATATATGCGGCCGCCTAGAGCCCCAGGTATTCAGC	The hisoQC that comprise terminator codon of amplification for inserting pcDNA 3.1
			EGFP-1 (SEQ ID NO：85)	ATATCTCGAGTCCATCGCCACCATGGTGAGC	Amplification EGFP

Primer	Sequence 5 ' → 3 '	Application
			EGFP-2 (SEQ ID NO：86)	ATATCTCGAGTTACTTGTACA GCTCGTCCAT	Amplification EGFP
hisoQC SS EGFP pcDNA as (SEQ ID NO：87)	ATATGCGGCCGCATGTCGACGCTCCAAATGGTGTAG AACGC	Amplification hisoQC amino terminal sequence
			hQC C-FLAG pcDNA as (SEQ ID NO：88)	ATATGCGGCCGCTTACTTGTCATCGTCATCCTTGTAA TCCAAATGAAGATATTCCAA	Amplification hQC C-FLAG
hisoQC C-FLAG pcDNA as (SEQ ID NO：89)	ATATGCGGCCGCCTACTTGTCATCGTCATCCTTGTA ATCGAGCCCCAGGTATTCAGC	Amplification h-isoQC C-Flag
			Hs_QPCT_1_SG	QuantiTect primer Assay (200), Qiagen, Hilden	qPCR hQC
Hs_QPCTL_1_SG	QuantiTect primer Assay (200), Qiagen, Hilden	qPCR h-isoQC
			CCL2-F (SEQ ID NO：90) CCL2-R (SEQ ID NO：91)	GCCTCCAGCATGAAAGTCTC CAGATCTCCTTGGCCACAAT	qPCR CCL2
CCL7-F (SEQ ID NO：92) CCL7-R (SEQ ID NO：93)	ATGAAAGCCTCTGCAGCACT TGGCTACTGGTGGTCCTTCT	qPCR CCL7
			CCL8-F (SEQ ID NO：94) CCL8-R (SEQ ID NO：95)	TCACCTGCTGCTTTAACGTG ATCCCTGACCCATCTCTCCT	qPCR CCL8
CCL13-F (SEQ ID NO：96) CCL13-R (SEQ ID NO：97)	ATCTCCTTGCAGAGGCTGAA AGAAGAGGAGGCCAGAGGAG	qPCR CCL13
			HIF1α-F (SEQ ID NO：98) HIF1α-R (SEQ ID NO：99)	CACAGAAATGGCCTTGTGAA CCAAGCAGGTCATAGGTGGT	qPCR HIF1α
AIM1-F (SEQ ID NO：100)	TCCTTTCATCCTGGAACCTG	qPCR AIM1

Primer	Sequence 5 ' → 3 '	Application
			AIM1-R (SEQ ID NO：101)	CGCCTCTTCTGTTTCACCTC
AIM2-F (SEQ ID NO：102) AIM2-R (SEQ ID NO：103)	AAGCGCTGTTTGCCAGTTAT CACACGTGAGGCGCTATTTA	qPCR AIM2
			MAGEA1-F (SEQ ID NO：104) MAGEA1-R (SEQ ID NO：105)	GTCAACAGATCCTCCCCAGA CAGCATTTCTGCCTTTGTGA	qPCR MAGEA1
MAGEA2-F (SEQ ID NO：106) MAGEA2-R (SEQ ID NO：107)	AGGTGGAGAGCCTGAGGAAT CTCGGGTCCTACTTGTCAGC	qPCR MAGEA2
			MAGEA10-F (SEQ ID NO：108) MAGEA 10-R (SEQ ID NO：109)	AAGCGAGGTTCTCGTTCTGA TGACCTCTTGCTCTCCCTGT	qPCR MAGEA10
MAGEB2-F (SEQ ID NO：110) MAGEB2-R (SEQ ID NO：111)	CTTCAAGCTCTCCTGCTGCT CGACCCTGACTTCCTGGTTA	qPCR MAGEB2
			MART1-F (SEQ ID NO：112) MART1-R (SEQ ID NO：113)	GCTCATCGGCTGTTGGTATT ATAAGCAGGTGGAGCATTGG	qPCR MART1
MCL1-F (SEQ ID NO：114) MCL1-R (SEQ ID NO：115)	ATGCTTCGGAAACTGGACAT ATGGTTCGATGCAGCTTTCT	qPCR MCL1
			TYR-F (SEQ ID NO：116) TYR-R (SEQ ID NO：117)	TACGGCGTAATCCTGGAAAC ATTGTGCATGCTGCTTTGAG	qPCR TYR
TYRP1-F	CCGAAACACAGTGGAAGGTT	qPCR TYRP1

Primer	Sequence 5 ' → 3 '	Application
			(SEQ ID NO：118) TYRP1-R (SEQ ID NO：119)	TCTGTGAAGGTGTGCAGGAG
TYRP2-F (SEQ ID NO：120) TYRP2-R (SEQ ID NO：121)	GGTTCCTTTCTTCCCTCCAG AACCAAAGCCACCAGTGTTC	qPCR TYRP2

Table 5: the purifying of GST-isoQC fusion rotein after expression in escherichia coli.The fusion rotein of purifying is for measuring QC activity.

Purification step	1	2	3	4
					Method	Ni ²⁺-IMAC(EBA)	GST label A C	GF (desalination)	IEX(UNO S)
Post type (Amersham Biosciences AB.Sweden)	Sequestering Sepharose Fast Flow	Glutathione S epharose 4 Fast Flow	Sephadex G-25 Fine	" successive bed " matrix BIO-Rad
					Column dimension	d＝2.5cm l＝42cm CV＝206cm ³	d＝1.6cm 1＝10cm CV＝20cm ³	d＝2.6cm l＝10cm CV＝53cm ³	d＝1.2cm l＝5.3cm CV＝6cm ³
Equilibrium process damping fluid/agent pH volume	PBS 7.3 10CV	PBS 7.3 10CV	25mMMes 6.0 10CV	25mM Mes 6.0 10CV
					Middle (washing) pH of buffer volume	PBS 0.5mM Histidine 7.3 10CV	PBS 7.3 10CV	-	25mM Mes 6.0 10CV
Elution buffer pH volume	PBS 100mM Histidine 7.3 1.5CV	50mM Tris 10mM Triptide (reduced form) 8.0 (reverse direction flow)	25mMMes 6.0 1CV	25mM Mes gradient elution NaCl 6.0 CV

Table 6: imdazole derivatives is to the K of the competitive inhibition of people QC and people isoQC ₁-value.People isoQC expresses in e. coli bl21 (hisoQCdt) or pichia pastoris phaff (YSShisoQC).

Inhibitor	Ki(μM)hisoQCdt	Ki(μM)YSShisoQC	Ki(μM)hQC
				Imidazoles	220±1	235±13	103±2
Benzoglyoxaline	200±8	250±5	138±4
				1-benzyl imidazole	7.3±0.5	6.2±0.2	7.1±0.1
1-Methylimidazole	80±5	82±3	39.7±0.2
				PBD150 1-(3,4-dimethoxy-phenylf)-3-(3-imidazoles-1-base-propyl group)-thiocarbamide	0.48±0.03	0.519±0.001	0.0584±0.0002

Embodiment 6: people isoQC expression and purification in pichia pastoris phaff

host strain and substratum

Use coli strain DH5 α with breed plasmid and pichia pastoris phaff strain X-33 for expressing people isoQC in yeast.E. coli strains and pichia pastoris phaff strain are cultivated according to manufacturer specification (Qiagen (DH5 α), invitrogen (X-33)), transformed and analyze.Recommend according to manufacturers, prepare the substratum required for intestinal bacteria and Luria-Bertani (LB) substratum.As described in pichia spp handbook (invitrogen, catalog number (Cat.No.) K1740-01), preparing substratum required for pichia pastoris phaff and BMMY, BMGY, YPD, YPDS and getting out the concentration of microbiotic and Zeocin.This handbook also comprises the whole associated description for operating yeast.

the molecular cloning of the plasmid vector of encoding human QC

Whole cloning process uses standard molecular biological technique to carry out.For expressing in pichia pastoris phaff X-33, use pPiCZ α A (Invitrogen).The cDNA starting from the one-tenth acquaintance isoQC of the 30th bit codon (counting from methionine(Met) II) merges with the plasmid meeting open reading-frame (ORF) mode and coding for alpha-factor, and wherein said α-factor instructs protein to enter Secretory Pathway.After utilizing primer hisoQCHISC-TermpPICZAA-1 (SEQIDNO:62) or hisoQCHISN-TermpPICZAA-1 (SEQIDNO:63) to increase as antisense primer as sense primer and hisoQCHISN-TermpPICZAA-2 (SEQIDNO:64) and hisoQCHISC-TermpPICZAA-2 (SEQIDNO:66) (table 4), utilize restriction site NotI and EcoRI, described fragment is inserted described expression vector.Depend on described construct, in the 55th (Ile) and the 351st (Cys) codon, import sudden change.This mutagenesis is carried out according to standard PCR amplification techniques, uses DpnI (changing the site-directed mutagenesis kit of II fast, Stratagene, catalog number (Cat.No.) 200524) digestion parental DNA subsequently.Schematically show the construct produced in fig. 17.

the conversion of pichia pastoris phaff and expression on a small scale

1-2 μ g plasmid DNA is used for according to manufacturer specification (BioRad) by electroporation transformed competence colibacillus P. pastoris cell.Selecting containing on the flat board of 100 μ g/mlZeocin.For checking recombination yeast clone when isQC expresses, recombinant chou is cultivated 24 hours in the 10ml tapered tube containing 2mlBMGY.After this, yeast is centrifugal and be resuspended in the 2mlBMMY containing 0.5% methyl alcohol.Described concentration 72 hours are maintained by adding methyl alcohol every 24 hours.Subsequently, the QC measured in supernatant liquor is active.The most highly active clone of performance is selected to be used for further experiment and fermentation.According to expressed construct, the isoQC-in substratum active different (Figure 18).

hisoQC is at the expression and purification of pichia pastoris phaff

For expressing isoQC on a large scale in pichia pastoris phaff, keep the condition as described in expression on a small scale, but cumulative volume is 8L.Express and carry out in shaking flask.After expression, cell passes through centrifugal (1500xg, 20 minutes) and is separated from substratum and abandons throw out.The pH-value of supernatant liquor is adjusted to neutrality, recentrifuge for the first purification step.Utilize 3-step scheme purifying (table 7).Purity analyzes display by the SDS-PAGE in Figure 19.

Table 7: the purifying of hisoQC (YSShisoQCN55IC351AC-His) after expressing in pichia pastoris phaff.The fusion rotein of purifying is used for determining QC activity and pH dependency.

Purification step	1	2	3
				Method	Ni ²⁺-IMAC	HIC	GF (desalination)
Post type (Amersham Biosciences AB, Sweden)	Sequestering Sepharose Fast Flow	Butyl Sepharose 4 Fast Flow	Sephadex G-25 Fine
				Column dimension	d＝2.5cm l＝42cm CV＝206cm ³	d＝1.6cm l＝15.5cm CV＝23cm ³	d＝2.6cm l＝10cm CV＝53cm ³
Equilibrium process pH of buffer volume	50mM NaH ₂PO ₄ 7.0 10CV	30mM NaH ₂PO ₄ 1M(NH ₄) ₂SO ₄ 7.0 10CV	50mM Bis-Tris 100mM NaCl 6.8 10CV
				Middle (washing) pH of buffer volume	50mM NaH ₂PO ₄0.5mM Histidine 7.0 10CV	30mM NaH ₂PO ₄ 1M(NH ₄) ₂SO ₄ 7.0 6CV	-
Elution buffer pH volume	50mM NaH ₂PO ₄100mM Histidine 7.0 1.5CV	30mM NaH ₂PO ₄ 7.0 5CV	50mM Bis-Tris 100mM NaCl 6.8 1CV

Result

People isoQC successfully expresses in methylotrophic yeast pichia pastoris phaff.Create several different construct, to select the optimum condition of the expression (Figure 17) in yeast.As shown in Figure 18, be expressed and the QC existed in the substratum of expressivity cell activity change according to expressed construct.The importing of glycosylation site causes correct secretion, as observed from construct YSShisoQCN55IC351AC-His and YSShisoQCN55IC-His.In view of most high reactivity in the medium, construct YSShisoQCN55IC351AC-His is expressed on a large scale and purifying.Implement as described in table 7, purification yield is 59%.Apparent homogeneous protein is glycosylated, as shown by the displacement (Figure 19) of mobility to lower molecular weight.Glycosylation does not affect the catalytic activity of enzyme.

The pH dependency of embodiment 7:hisoQC

The fluorometry of (describing in embodiment 5) H-Gln-β NA is used to study the specific pH dependency of catalysis.With the concentration of substrate of 7 μMs (namely at [S] < < K _m) react.Therefore, the specificity constant observed can directly be derived from the initial velocity of substrate conversion progress curve.In these researchs, by using, HCl or NaOH is adjusted to the 0.075M acetic acid of required pH to reaction buffer, 0.075MMes and 0.15MTris forms.The constant ionic strengths within the scope of extremely wide pH guaranteed by this damping fluid.Use the enzyme kinetics data that following equation evaluation obtains:

K _cat/ K _m(pH)=k _cat/ K _m(boundary value) * 1/ (1+ [H ⁺]/K _hS+ K _e1/ [H ⁺]+K _e1/ [H ⁺] * K _e2/ [H ⁺]),

Wherein k _cat/ K _m(pH) (observing) pH-dependency kinetic parameter is referred to.K _cat/ K _m(boundary value) refers to pH dependent/non-dependent (" restricted ") value.K _hS, K _e1and K _e2refer to the dissociation constant of two groups that dissociate in dissociate group and the described enzyme in acid pH range respectively.Use GraFit software (for the 5.0.4 version of windows, ERITHACUSSOFTWARELtd., Horley, UK) to evaluate the enzyme kinetics data obtained, evaluation is carried out to whole dynamics data.

Result

HisoQC shows specific optimal pH on pH7-8.Therefore, the optimal pH of catalysis and people QC fairly similar.Described data then cause the dependent good explanation (Figure 22) of the pH of hisoQC and hQC according to the matching of the model based on three groups that dissociate.Therefore, the catalysis of two kinds of enzyme reactions affects by the similar group that dissociates, and shows similar catalyst mechanism on the whole.

Determined pKa-value displayed in Table 8.Obviously to see between hisoQC and hQC only pKa difference significantly.In hQC, this pKa corresponds to the pKa of substrate dissociation constant.Between hQC and hisoQC, imperceptible difference may cause because of the constructive variations (induced-fit) occurred in isoQC catalytic process, thus affects pH dependency.

Embodiment 8: to the research of glutamyl cyclase activity

Describe people QC enzyme catalysis aminoterminal L-glutamic acid and be cyclized into Pyrrolidonecarboxylic acid.Therefore, QC participates in the generation of the 4 amyloid that pGlu modifies.

For studying the cyclisation of L-glutamic acid, Purification of Human QC and people isoQC and monitor amyloid A β (3-11) [pGlu-A β (3-11)] that pGlu the modifies formation from A β (3-11).Reaction is by 20 μ l substrates (the 2.5mM stock solution in the 50mMMes damping fluid of A β (3-11), pH6.5) and 80 μ l enzyme (0.62mg/mlhQC stock solutions; 0.61mg/mlhisoQC stock solution in the 50mMMes damping fluid of pH6.5) composition.After 0 hour, 6 hours, 24 hours, 48 hours and 72 hours, take out sample (15 μ l) and boiled 5 minutes with termination reaction.By Maldi-Tof analytical reagent composition substrate conversion.Substrate and product differ 18Da in its molecular mass, the quality of the water discharged during this is cyclisation.

As shown in Figure 23, people QC and people isoQC (YSShisoQCI55NC351AC-His) catalysis A β (3-11) change into pGlu-A β (3-11).But based on the same protein concentration in two samples, can reach a conclusion: compared with hQC, the conversion of hisoQC to aminoterminal L-glutamic acid is much slow.Therefore, the lower specificity constant of glutamyl substrate conversion is also observed with glutamyl substrate.Cyclic action (Schilling, S. etc., 2004FEBSLett.563,191-196) is not observed under these conditions with the enzyme of deactivation.

Embodiment 9: the tissue specificity of mouse isoQC

Quantitative real time pcr is used to study the tissue distribution of mouse QC and mouse isoQC.From several Different Organs with tissue in be separated cDNA before, (isoQCmMetIs (SEQIDNO:68), isoQCmMetIas (SEQIDNO:69) (table 4) are separated mouse isoQC open reading-frame (ORF), and this open reading-frame (ORF) is derived from the chromosome coding district of mouse isoQC to use specific primers.

Described open reading-frame (ORF) is cloned into carrier pPCR-ScriptCAMSK (+) (PCR-ScriptCAM Cloning Kit, Stratagene) and the positive control be used as in PCR in real time assay method for the preparation of the typical curve under condition determination.Use the cDNA from 3-6 monthly age mouse, realize the tissue-specific sign that misoQC is expressed.RNA separating kit (Macherey with Nagel) is used to be separated total serum IgE from 30mg tissue.RNA concentration and purity are by gel electrophoresis (sepharose) and spectrophotometry assessment.For synthesis cDNA, use 1 μ gRNA.Recommend according to supplier, use reversed transcriptive enzyme SuperscriptIIRT (Invitrogen) to react, described cDNA is stored in-80 DEG C.

Use " LightCycler " (Corbettreserach), utilize the transcript concentration in the quantitative analysis different tissues of " QuantiTectSYBRGreenPCR " (Qiagen).DNA standard (the mouse cDNAisoQC of clone) is for quantitatively.Copy number is calculated: (X according to following equation ^g/ _{μ l}dNA)/(plasmid length bp*660) * 6.022*1023=Y ^molecule/ _{μ l}.Described DNA standard contains 10 ⁷-10 ^{1 molecule}/ _{μ l}4 concentration of scope, and threshold concentration (10 °).Reaction scheme displayed in Table 8.Show result in fig. 24.

Use mQCRT aminoterminal (SEQIDNO:73) and mQCRT aminoterminal (SEQIDNO:74) as primer, use same approach with the mouse QC that increases.

Table 8: the quantitative real-time PCR reactions scheme using Roto-GeneRG3000 (Corbettreserach)

Result

As shown in Figure 24, mouse QC and mouse isoQC expresses in whole organs of inspection.Contrary with mouse QC, the variation that between Different Organs, mouse isoQC expresses is less, shows the lower severity of transcriptional regulation.The data that mQC expresses are with previously corresponding to the analysis of ox QC, and wherein use Northern-Blot analyzes described ox QC 1991ProcNatlAcadSciUSA88,10059-10063 such as () Pohl, T..In thalamus, hippocampus and cortex, observe QC expresses the highest.Therefore, QC expresses and mainly detects in neuronal tissue.In peripheral organ is as spleen and kidney, seldom detect that QC expresses.MisoQC also expresses in neuronal tissue, but is in lower level compared with mQC.On the contrary, very similar between the expression level of isoQC and QC in peripheral organ.

Based on the result of transcript concentration, reach a conclusion: associating active (isoQC and QC) should be the highest in brain.Therefore, the highest QC-protein level is present in the organ attacked as alzheimer's disease, Familial British Dementia and Familial Danish Dementia by amyloidosis.

Embodiment 10: suppress people isoQC by heterocycle sequestrant

result

Previous use heterocycle sequestrant as 1,10-phenanthroline and dipicolinic acid have studied to the QC of different sources time m-dependency restraining effect (6,9).Similarly, also time-dependent manner ground is by heterocycle sequestrant 1,10-phenanthroline (Figure 25) and the deactivation of dipicolinic acid's (not shown) for h-isoQC, and this is clear points out that metal relies on activity.In addition, EDTA also suppresses h-isoQC (Figure 25).This and QC form sharp contrast, because EDTA causes noticeable suppression all to people QC, pig QC or mouse QC.But the even more high inhibition of EDTA to hisoQC shows to there is metal-dependant catalysis.

Embodiment 11: the Subcellular Localization using the hisoQC of cellular fractioning research

cellular fractioning

Next day after transfection, expressivity HEK293 cell D-PBS is washed and passes through to collect upper centrifugal 5 minutes of 500 × g at 4 DEG C.Subsequently, abandon D-PBS and cell be resuspended in 1ml Disruption buffer (50mMTris, 50mMKCl, 5mMEDTA, 2mMMgCl ₂, be adjusted to pH7.6 with HCl) in and be broken by 30 squeezings in Potter cell homogenates machine.Suspension 4 DEG C at 700 × g upper centrifugal 10 minutes.The precipitation obtained to be resuspended in 300 μ l Disruption buffer and called after relic fraction (D).The supernatant liquor of gained further at 4 DEG C at 20,000 × g upper centrifugal 30 minutes.Precipitation display is attached most importance to film fraction (HM) and being resuspended in 200 μ l Disruption buffer.The supernatant liquor of gained uses ultracentrifuge (Beckmann) at 4 DEG C at 100,000 × g upper centrifugal 1 hour.The precipitation obtained to be resuspended in 200 μ l Disruption buffer and to be called light film fraction (LM).By supernatant liquor called after soluble fraction (S).By relic fraction, heavy film fraction and light film fraction supersound process 10 minutes.Bradford method is used to measure the protein content of whole fraction.Subsequently, analyze the QC activity of fraction and use Western blotting to dye to the labelled protein of fraction.

result

For further confirmation, biochemical analysis is carried out to the QC activity distribution expressed derived from hisoQC and hQC.Natural hisoQC and hQC starting from methionine(Met) I and II expresses respectively in HEK293 cell.After cell grade is separated, uses fluorometry, utilize H-Gln-β NA as substrate, the QC measured in often kind of fraction is active.In cell with empty carrier (pcDNA) transfection, almost measure less than specificity QC active.When expressing natural hisoQC (MetI) and hisoQC (MetII), can detect that QC is active easily, and most high reactivity is present in weight film fraction (MetI:40 ± 2 μm ole/ minute/g; MetII:36 ± 1.5 μm ole/ minute/g) and substratum (MetI:30 ± 2 μm ole/ minute/g; MetII:54 ± 3 μm ole/ minute/g) in.On the contrary, it is active that hQC shows most high specific QC in substratum (1339 ± 76 μm of ole/ minute/g), subsequently display time high specific QC activity (Figure 26 A) in heavy film fraction (251 ± 21 μm of ole/ minute/g).

In addition, calculate absolute activity, show that the expression of hisoQC (MetI) and hisoQC (MetII) mainly causes QC activity in born of the same parents to increase, relic (MetI:1032 ± 9nM/ minute in sight; MetII:1110 ± 10nM/ minute) and heavy film fraction (MetI:374 ± 20nM/ minute; MetII:281 ± 12nM/ minute) increase of middle QC activity.A small amount of QC activity (MetI:27 ± 2nM/ minute is only there is in substratum; MetII:53 ± 3nM/ minute).On the contrary, by hQC express derive QC activity in the medium (1138 ± 65nM/ minute) and in intracellular region room (relic: 1089 ± 14nM/ minute; Heavy film fraction: 583 ± 38nM/ minute) display high reactivity, support the hisoQC Golgi localization (Figure 26 B) as passed through as shown in tissue chemical analysis.

The data obtained by expressing natural enzyme obtain having the hisoQC (MetI and MetII) of carboxyl terminal FLAG label and the support (Figure 26 C) of hQC by expressing further.Compare with mitochondrial labelled protein with Golgi complex, the western blot analysis of the FLAG labeling protein of gained disclose hisoQC (MetI) and hisoQC (MetII) mainly at inner cellular localization in described relic fraction and weight film fraction, and hQC is enriched in substratum, but be also present in described relic fraction and heavy film fraction.Golgi complex and mitochondrial labelled protein video picture are shown that these compartments are present in described relic fraction and heavy film fraction.In addition, 65kDa mitochondrial protein also exists in soluble fractions with smaller portions.

Embodiment 12: to the analysis of hisoQC Golgi retention signal

For understanding fully when the aminoterminal transbilayer helix of prediction is responsible for being detained hisoQC in Golgi complex, by the signal peptide comprising described transbilayer helix starting from MetI and MetII place to meet open reading-frame (ORF) mode and EGFP clones.Gained carrier hisoQC (MetI) SSEGFP and hisoQC (MetII) SSEGFP is at LN405 cells and use confocal laser scanning microscope, CLSM to check in the mode being similar to total length hisoQCEGFP fusion rotein.The expression of hisoQC (MetI) SSEGFP causes as located the viewed identical Golgi complex of total length hisoQC (MetI) EGFP fusion rotein.Again, hisoQC (MetI) SSEGFP is not observed to mitochondrial transport (Figure 27 A).In addition, the expression of amino-terminal truncated version peptide hisoQC (MetII) SSEGFP also causes this protein-enriched in Golgi complex.Similar with hisoQC (MetI) SSEGFP, plastosome EGFP fluorescence (Figure 27 B) can not be recorded to.Therefore, the amino terminal sequence of hisoQC causes this protein be transported to ER film with common interpretative system and cause being trapped in Golgi complex.In addition, because hisoQC (MetII) SSEGFP expresses, Golgi retention signal is roughly located be between the 19th methionine(Met) and Serine the 53rd (from MetI counting amino acid).

Extra topological Analysis discloses the possibility of hisoQC aminoterminal and glycosyltransferase function homology.Glycosyltransferase is II type transmembrane protein, has short endochylema sequence, catalyst structure domain in follow-up transbilayer helix and a large chamber.Obviously, this structural domain structure constructs substantially the same (Figure 28) with the structural domain found misoQC with hisoQC.For numerous glycosyltransferase, determine Golgi retention signal and be positioned at described membrane spaning domain.In addition, for some enzyme in these enzymes, find that the endochylema sequence described in brachymemma does not affect activity or the location of this protein.In a word, provide such evidence, namely hisoQC is II type transmembrane protein, shows in the Golgi complex similar to glycosyltransferase and is detained.

Embodiment 12: detect the QPCTLmRNA in different people cancerous cell line and tissue

qPCR analyzes

Substantially as described in example 9 above, quantitative PCR in real time (qPCR) technology is used to carry out the analysis expressed people QPCTL in human carcinoma cell line.For surveying QPCTLmRNA, use the primer of primer assay method, this primer covers for comprising exon/exon 1 to get rid of the coamplification of genomic dna.Recommend to carry out QPCR according to manufacturers.In table 9, reaction mixture is described and PCR program displayed in Table 8.

The composition of table 9:qPCR mixture

Composition	Volume (μ l)
		2x QuantiTect SYBR Green PCR Master Mix(2,5mM MgCl ₂)	7.5
10x QuantiTect primer assay method	1.5
		CDNA (≤100ng/ reaction)	1
Pure water	5

Use " LightCycler " (Corbettreserach), utilize the transcript concentration in the quantitative analysis different tissues of " QuantiTectSYBRGreenPCR " (Qiagen).DNA standard (the cDNAisoQC mouse of clone) is for quantitatively.Copy number is calculated: (X according to following equation ^g/ _{μ l}dNA)/(plasmid length bp*660) * 6.022*1023=Y ^molecule/ _{μ l}.Described DNA standard contains 10 ⁷-10 ¹4 concentration of molecule/μ l scope, and threshold concentration (10 °).

Rotor-Gene function software (Corbettreserach) is used to evaluate the result of qPCR.

result

the expression of QPCTL in different carcinoma clone

In checked cancerous cell line, human melanoma cell shows the most high expression level (about 7000 copy/50ng total serum IgE) of QPCTL transcript, simultaneously the minimum expression (365 copy/50ng total serum IgE) of people's soft tissue sarcoma cell system display QPCTL.Carcinoma of the pancreas shows 2100 copies, and thyroid carcinoma shows 3500 copies, and cancer of the stomach has 4100 copies (median) (Figure 29).

the expression of QPCTL in different melanoma cell series

Recently confirmed that melanoma cells has higher QPCT and expresses (Gillis, J.S., J.Transl.Med.4 (2006), 4:27).Therefore, the QPCTL analyzed in different melanoma cell series expresses.As shown in Figure 30, in whole melanoma cell series, all detect that QPCTL expresses.From 2025 Mel_ZL_11 system, a copy/50ng total serum IgE is changed to 18043 copy/50ng total serum IgE in Mel_ZL12 system in variation between clone.

The dependency of table 10:QPCT and QPCTL and tumor associated antigen (taa) and QPCT and QPCTL dependency each other.

In addition, QPCT with QPCTL expresses relevant to the expression of tumor associated antigen (taa).Melanoma-Specific tumor associated antigen is selected by database retrieval and the result delivered.Wherein, AIM1 and AIM2 (AbsentInMelanoma (lacking in melanoma)), MAGEA1-A2 ,-A10 and MAGEB2 (melanoma antigen family A and B), MART1 (melanoma antigen of T-cell recognition), TYR (tyrosine oxidase), TYRP1 and TYRP2 (tyrosinase-related protein) and MCL-1 (myelocytic leukemia) are the tumor associated antigens in melanoma.Use SPSS statistical software comparative data.

Dependency between QPCT and MAGEB2 is significant (p=0.0436).In addition, the dependency between QPCT and MART1 (p=0.002), QPCTL and MART1 (p=0.008) and QPCT and TYR (p=0.0023) is also statistics highly significant.Described dependency shows direct dependency, and it is higher that this shows that QPCT/QPCTL expresses, and tumor associated antigen is expressed higher.Sole exception is the dependency between TYR and MCL1, and it shows indirect dependence.

the expression of QPCT and QPCTL in different tumor tissues

The expression of QPCT and QPCTL is evaluated in the tumor tissues of soft tissue sarcoma, cancer of the stomach and thyroid carcinoma.Finding that QPCT expresses the highest in thyroid carcinoma, is secondly cancer of the stomach and soft tissue cancer (table 11).QPCTL is expressed and also observes identical sequence, but, the copy number of QPCTL transcript always lower than to the viewed copy number of QPCT transcript, as Student ' st-checks (p _{soft tissue sarcoma}=0.001; p _{cancer of the stomach}=4.8E-7; p _{thyroid carcinoma}=0.04) (table 11; Figure 31).

The comparison that table 11:QPCT and QPCTL expresses in different tumor tissues

	Soft tissue sarcoma's (119 increment product)	Cancer of the stomach (47 increment product)	Thyroid carcinoma (29 increment product)
				QPCT	1293	2985	8303
QPCTL	170	469	2540

Pearson's bilateral significant correlation in cancer (p=2E-31) and cancer of the stomach (p=0.015) is organized to the further research announcement of QPCT and QPCTL expression level.Dependency (p=0.46) is not observed to QPCT and the QPCTL expression level in thyroid carcinoma.

qPCTL relies on the expression of differentiation phase in cancer of the stomach

For cancer of the stomach, the QPCTL that have studied in the sample representing the different Tumor Differentiation phase expresses.Healthy tissues served as control around tumour.Healthy tissues compares with tumor tissues, and QPCTL significantly higher in display tumor tissues expresses (p=0.04).Undifferentiated cancer of the stomach display is expressed lower than the QPCTL of healthy tissues.Compared with healthy tissues, low differentiation and the good cancer of the stomach to medium differentiation do not show difference (Figure 32) in median.

the expression of QPCT and QPCTL in asynchronous thyroid carcinoma

Express with regard to QPCT and QPCTL, study asynchronous thyroid carcinoma.Be categorized as follicular thyroid carcinoma (FTC), papillary thyroid carcinoma (PTC) and undifferentiated thyroid carcinoma (UTC) according to the name of the World Health Organization (WHO) by stages.From the sample served as control of thyrocele patient.

QPCTmRNA level (median) is higher than (thyrocele: 2100 copy/50ng total serum IgE) in nonneoplastic tissue in the thyroid carcinoma FTC (6700 copy/50ng total serum IgE) and PTC (16000 copy/50ng total serum IgE) of differentiation.UTC has 5400 copy/50ng total serum IgE and higher than the copy number observed in thyrocele 2.5 times.The mRNA copy number of QPCT is significantly higher than thyrocele (p=0.04, Student ' st-inspection) (Figure 33) in whole thyroid tumor.

QPCTLmRNA level in thyroid carcinoma is uniform.Similar to thyrocele (2500 copy/50ng total serum IgE) with the sample of UTC (2500 copy/50ng total serum IgE) from FTC (2600 copy/50ng total serum IgE).The expression of QPCTL in PTC drops to 1900 copy/50ng total serum IgE (Figure 34) slightly.

In a word, QPCT and QPCTL expresses comparably in thyrocele.But in tumor tissues, the expression of QPCT increases, and the expression of QPCTL keeps stable.

Embodiment 13: the expression of QPCT and QPCTL in human cell line after research and different stimulated thing incubation

clone and substratum

Use human embryonic kidney cell line HEK293, people's acute monocytic leukemia clone THP-1 and follicular thyroid carcinoma clone FTC-133 carry out stimulation test.Cell appropriate media (DMEM, 10%FBS be used for HEK293, RPMI1640,10%FBS be used for THP-1 and DMEM/F12,10%FBS be used for FTC-133) in, at 37 DEG C and 5%CO in humidified atmosphere ₂lower cultivation.

use the stimulation of biologically active peptides, chemical or LPS

HEK293 and FTC-133 cell is cultivated as adherent culture thing and THP-1 cell is cultivated with suspended pattern.For stimulation assay method, by 2 × 10 ⁵it is dull and stereotyped that individual FTC-133 cell and HEK293 cell are transferred to 24 holes.When HEK293, dull and stereotyped with type i collagen bag by guarantee that appropriateness adheres to.In addition, by 2 × 10 ⁶individual THP-1 cell is cultivated in 24 hole suspension flat boards.Whole stimulation test carries out under serum-free condition.FTC-133 overnight culture.After this, other 24 hours of cell adapted serum free medium and start stimulation by conditioned medium is replaced with fresh serum-free media.HEK293 cell pellet overnight is cultivated and after this, is started the stimulation using respective substance without the need to adapting to serum-free condition, and reason is the HEK293 genetic morphology change of cultivating under serum-free condition more than 24 hours.THP-1 cell is seeded in the serum free medium containing respective substance.The stimulator applied and final concentration list in table 12.

Table 12: for studying in human cell line the stimulator regulating hQC and hisoQC

Title	Final concentration
		Butyric acid (BA)	2mM
PHGF (HGF)	10ng/ml
		Lipopolysaccharides (LPS)	1,10μg/ml
Transforming growth factor-beta (TGF β)	10,100ng/ml
		Tumor necrosis factor alpha (TNF α)	10,100ng/ml

Cell and corresponding stimulator incubation 24 hours.After this, Nucleo-is used rNAII test kit (Macherey-Nagel) from this cellular segregation total serum IgE and store until qPCR measure.

use the hormesis of hypoxemia

The cover plant of THP-1, HEK293 and FTC-133 cell difference is at 2 25cm ²in tissue culture plate.Thus a plate of often kind of clone serves as negative control, cultivate 24 hours under normal growing conditions.Another plate be placed in anoxic sack with anoxic reagent ( p, Merck) and indicator is together.By this pouch seal to guarantee isolated air conditions.Cell is also cultivated 24 hours and subsequently, uses Nucleo- rNAII test kit (Macherey-Nagel) is separated total serum IgE and stores until qPCR measures.

result

the basal expression of QPCT and QPCTL in HEK293, FTC-133 and THP-1

Evaluate in clone HEK293, FTC-133 and THP-1 used basal expression and be subsequent stimuli experiment get ready.The copy number of QPCT and QPCTL transcript is summed up in table 13.

Table 13: the basal expression of QPCT and QPCTL in different clone

the impact that selected stimulator is expressed QPCT and QPCTL

The modulability binding site not describing the promotor of QPCT and QPCTL so far and the signal transduction pathway causing them to regulate.Therefore, different clone and stimulator is used to implement stimulation test.QPCTmRNA in HEK293 cell stimulates horizontally through use TNF-α, HGF and butyric acid and increases.In addition, have studied the regulating effect to the CCL2 as QPCT/QPCTL substrate.TNF-α and butyric acid increase the amount of CCL2 transcript in HEK293.HGF expresses not impact to CCL2.On the contrary, QPCTL does not regulate (Figure 35) by TNF-α, HGF and butyric acid.

In addition, use LPS and TGF-β to stimulate FTC-133 and detect the regulating effect to QPCT, QPCTL and CCL2.In FTC-133, LPS and TGF-β stimulates QPCTmRNA to express, but does not induce QPCTL and CCL2 to express (Figure 36).

This experiment stimulates THP-1 cell to confirm further by use LPS (1 μ g/ml), LPS (10 μ g/ml), TGF-β and TNF-α.As FTC-133 and HEK293 is observed, use different stimulated thing, QPCT may be induced to express, in addition, use LPS and TNF-α to express induction of CCL2.Equally, fail to observe the induction of QPCTLmRNA or check (Figure 37).

In a word, this experiment is disclosed QPCT and can be regulated by group stimulator of in different clone (LPS, TNF-α, HGF, butyric acid and other).On the contrary, QPCTL may not check stimulator stimulation by institute or check, thus implies house keeper's function of QPCTL.

selected stimulator is on the impact of the expression of QPCT and substrate thereof

Induced by multiple stimulator because QPCT expresses, therefore propose such problem: whether QPCT induction occurs with conjunction QPCT substrate CCL2, CCL7, CCL8 together with the induction of CCL13.Therefore, use THP-1 monocyte, use LPS (1 μ g/ml) respectively, LPS (10 μ g/ml), TGF-β (100ng/ml) and TNF-α (100ng/ml) stimulate.THP-1 expresses whole chemokine with basal level, and this is important for comparing at negative control and irriate cell paper.

LPS and TNF-α causes reliably inducing the chemokine and QPCT all checked in THP-1 cell.TGF-β is so ineffective as stimulator, and QPCT, CCL2, CCL7 and CCL8 of induction 2 times at most express.CCL13 is checked (Figure 38) by TGF-β hormesis.

qPCT and QPCTL expresses to be stimulated by hypoxemia

QPCTL expresses and may not be regulated by chemical agent, biologically active peptides or LPS.Therefore, we check QPCTL to express by no by hypoxemia adjustment.As summed up in Figure 39, the expression of QPCTL is induced but not the expression of QPCT in hypoxia-selective ground.Comparatively speaking, hypoxic inducing factor-1 a (HIF1a) is thwarted and reaches 15% (Figure 39 A) and 45% (Figure 39 C).These data show contacting of QPCTL and hypoxemia.

The synthesis of inhibitor

Synthetic schemes 1: example 1-53, the synthesis of 96-102,136-137

Reagent and condition: (a) NaH, DMF, 4 hours, room temperature; (b) 8 hours, 100 DEG C; (c) H ₂n-NH ₂, EtOH, 8 hours, backflow, 4NHCl subsequently, 6 hours, backflow, (d) R ³-NCO, EtOH, 6 hours, backflow, (e) 3,4 dimethoxy-phenylf-lsothiocyanates.

Synthetic schemes 2: the synthesis of example 54-95

Reagent and condition: (a) R-NCS, EtOH, 6 hours, backflow; (b) WSCD, 1H-imidazoles-1-propylamine, DMF, 2 hours, room temperature.

Synthetic schemes 3: the synthesis of example 103-105

Reagent and condition: (a) NaH, DMF, room temperature, 3 hours; (b) LiAlH ₄， diox, backflow, 1 hour; C () R-NCS, EtOH, reflux 6 hours.

Synthetic schemes 4: the synthesis of example 106-109

Reagent and condition: (a) EtOH, 2 hours, backflow.

Synthetic schemes 5: the synthesis of example 110-112

Reagent and condition: (a) 1H-imidazoles-1-propylamine, triethylamine, toluene, 12 hours, backflow.

Synthetic schemes 6: the synthesis of example 113-132

Reagent and condition: (a) CAIBE, 1H-imidazoles-1-propylamine ， diox, 0 DEG C, 12 hours; (b) Lawesson reagent, EtOH, backflow, 8 hours.

Synthetic schemes 7: the synthesis of example 133-135

Reagent and condition: (a) 1H-imidazoles-1-propane acid chloride, CH ₂cl ₂,-10 DEG C, 1 hour; (b) Lawesson reagent ， diox, backflow, 8 hours.

Synthetic schemes 8: the synthesis of example 138

Reagent and condition: (a) EtOH, backflow, 8 hours.

Synthetic schemes 9: the synthesis of example 139

Reagent and condition: (a) 75% concentration H ₂sO ₄, 4 hours.

Synthetic schemes 10: the synthesis of example 140

Reagent and condition: (a) acetonitrile, reflux 2 hours.

Synthetic schemes 11: the synthesis of example 141

Reagent and condition: (a) NaH, DMF, 4 hours, room temperature; (b) 8 hours, 100 DEG C; (c) H ₂n-NH ₂, EtOH, 8 hours, backflow, 4NHCl subsequently, 6 hours, backflow, (d) 3,4 dimethoxy-phenylf-lsothiocyanates, EtOH, 6 hours, backflow.

Analysis condition

ESI-mass spectrum is obtained with SCIEXAPI365 spectrometer (PerkinElmer).Use DMSO-D ₆as solvent, record on BRUKERAC500 ¹h-NMR (500MHz) data.Chemical shift is expressed as relative to tetramethylsilane ppm (ppm) to low field displacement.Schizotype name is as follows: s (unimodal), d (bimodal), dd (bimodal is bimodal), t (triplet), m (multiplet) and br (broad signal).

Detailed synthesis describes

Example 1-12 and 14-53

1H-imidazoles-1-propylamine reacts 8 hours under reflux to the corresponding lsothiocyanates in ethanol.After this, except desolventizing remaining oil is dissolved in methylene dichloride.Organic layer NaHCO ₃saturated solution, NaHSO ₄saturated solution and salt solution wash twice, drying is also evaporated subsequently.By remaining solid recrystallize from ethyl acetate, produce this example thiocarbamide with yield 80-98%.

Example 13

1-(3-(1H-imidazoles-1-base) propyl group)-3-(3,4-Dimethoxyphenyl) thiocarbamide

4.0mmol isothiocyanic acid 3,4-dimethoxy phenyl ester and 4.0mmol3-(1H-imidazoles-1-base) alkyl-1-amine solvent are in 10mL dehydrated alcohol.Stir under reflux after 2 hours, described solvent is evaporated and the solid of gained recrystallize from ethanol.

Yield: 0.66g (51.3%); Fusing point: 160.0-161.0 DEG C

¹HNMRδ1.8-2.0(m，2H)，3.4-3.5(m，2H)，3.75(s，6H)，3.9-4.0(m，2H)，6.7-6.8(m，1H)，6.9(brm，2H)，6.95(s，1H)，7.15(s，1H)，7.55(brs，1H)，7.6(s，1H)，9.3(s，1H)；MSm/z321.2(M+H)，253.3(M-C3H ₃N ₂·)

Example 96-102

1H-imidazoles-1-propylamine reacts 8 hours under reflux to the corresponding lsothiocyanates in ethanol.After this, except desolventizing remaining oil is dissolved in methylene dichloride.Organic layer NaHCO ₃saturated solution, NaHSO ₄saturated solution and salt solution wash twice, drying is also evaporated subsequently.By remaining solid recrystallize from ethyl acetate, produce this example urea with yield 85-90%.

Example 136,137

1H-imidazoles-1-alkylamine is prepared according to document and is carried out hydrazinolysis subsequently from ω-bromo-alkyl-phthalimide and imidazole salts.Products therefrom is changed into the thiocarbamide of example 1-53, produce 88% (example 136) and 95% (example 137) yield.

Example 54-95

Whole institutes example by mixing room temperature reaction 2 hours and producing with the water-soluble carbodiimide (WSCD) in anhydrous dimethyl formamide and 1H-imidazoles-1-propylamine, producing three with yield 40-87% and replacing guanidine classes from corresponding thiocarbamide.

Example 103-105

Utilize 1 equivalent NaH, the 2-bromomethylphenyl cyanogen making imidazoles corresponding to DMF, room temperature reaction 3 hours, produces 1H-imidazoles-1-aminomethyl phenyl cyanogen.Remove described solvent and the oil of gained is heavily dissolved in diox.Use 1 equivalent LiAlH ₄, in corresponding amine, transform described prussiate.At interpolation KHSO ₄after saturated solution, diox is evaporated and passes through CHCl ₃extract waterbearing stratum.Vacuum concentration organic layer and being transformed in the corresponding thiocarbamide of example 1-53 by described amine, produces 78% (example 103) and 65% (example 104) and 81% (example 105) yield.

Example 106-109

From corresponding methanesulfonates-2-methyl-propyl-phthalimide, as described the amine in example 136-137, synthesize described amine.Products therefrom is changed into the thiocarbamide of example 1-53, produce example 106-109 with total recovery 25-30%.

Example 110-112

1H-imidazoles-1-propylamine 2-the chlorobenzene corresponding to toluene also [d] thiazole reacts 24 hours temperature 130 DEG C.Removing desolventizing and from methyl alcohol after recrystallize, producing example 110-112 with the amount of 55-65%.

Example 113-118,120-124 and 126-132

By adding CAIBE and the N-methylmorpholine of 1 equivalent, 1H-imidazoles-1-propylamine is reacted temperature 0 DEG C to without 2-phenylacetic acid corresponding in water diox.After 2 hours, make mixture heat to room temperature and stir 12 hours.After removal of the solvent, the oil of gained to be heavily dissolved in methylene dichloride and organic layer NaHCO ₃the aqueous solution and water washing, drying solvent is evaporated.Remaining oil is dissolved in and adds in Lawesson reagent diox.In stirring after 12 hours, add NaHCO ₃saturated solution, extracts waterbearing stratum by ethyl acetate Jiang diox evaporation, is separated by organic layer, dry and evaporated by solvent.Remaining solid crystallization from acetylacetic ester/ether, produces 113-118,120-124 and 126-132 with total recovery 62-85%.

Example 119

N-(3-(1H-imidazoles-1-base) propyl group)-2-(3,4-Dimethoxyphenyl) second sulphamide

4.0mmol triethylamine and 4.0mmol3-(1H-imidazoles-1-base) mixture of alkyl-1-amine in 20mL diox are dropwise added into the solution of the ice-cold stirring of 4.0mmol2-in 30mL diox (3,4-Dimethoxyphenyl) Acetyl Chloride 98Min..This mixture is made to heat to room temperature and stir 1 hour subsequently.After removing desolventizing by decompression method, resistates is heavily dissolved in 50mL methylene dichloride.Organic layer passes through 30mLNaHCO ₃saturated aqueous solution and water washing.Organic solution is dry, filtration, and solvent is under reduced pressure removed.After 50mL heavily dissolves in Shui oxane, add 2.2mmolLawesson reagent, and this mixture is heated to 90 DEG C and stirs 8 hours.By removal of solvent under reduced pressure, resistates is heavily dissolved in 50mL methylene dichloride.Organic layer passes through NaHCO ₃saturated aqueous solution washs three times, washes three times subsequently with water, dry, filters and removes organic solvent subsequently.Use centrifugal force-chromatographic apparatus (HarrisonResearchLtd.), utilize silica gel plate and the CHCl of layer thickness 2mm ₃/ MeOH gradient as elution system, by chromatography by compound purifying.

Yield: 0.14g (10.6%); Fusing point: 148.0-150.0 DEG C

¹HNMRδ2.0-2.15(brm，2H)，3.4-3.5(m，2H)，3.7(s，6H)，6.75-6.8(m，2H)，4.1-4.2(m，2H)，6.8-6.9(m，2H)，6.95-7.0(m，1H)，7.4(s，1H)，7.75-7.85(brm，1H)，8.6(s，1H)，10.2(s，1H)；MSm/z320.2(M+H)，252.2(M-C3H ₃N ₂·)

Example 125

N-(3-(1H-imidazoles-1-base) propyl group)-1-(3,4-Dimethoxyphenyl) cyclopropyl first sulphamide

11.06mmol3,4-Dimethoxyphenyl acetonitrile, the bromo-1-chloroethanol of 34.8mmol2-and 1.16mmol tri-chlorination Ethylbenzyl ammonium are dissolved in the 10mLKOH aqueous solution (60%).This mixture to be transferred in ultrasonic cleaner and room temperature vigorous stirring 3 hours.By the suspension of gained with the dilution of 40mL water and with 20mL dichloromethane extraction three times.The organic layer salt aqueous acid (1N) merged is washed, through Na ₂sO ₄dry and under elevated pressure except desolventizing.Use silica gel and use the oil that ethyl acetate/heptane is left by flash chromatography as elution system, producing 0.81g (34.4%) 1-(3,4-Dimethoxyphenyl) cyclopropyl formonitrile HCN.

3.9mmol1-(3,4-Dimethoxyphenyl) cyclopropyl formonitrile HCN and 11.2mmolKOH are suspended in 80mL ethylene glycol.This mixture stirs 12 hours under reflux.Subsequently, add 80mL water and extract waterbearing stratum 2 times with ether.After use HCl (1N) adjust ph to pH=4-5, extract waterbearing stratum three times with ether, subsequently by the organic layer that merges through Na ₂sO ₄dry also except desolventizing, produce 1-(3, the 4-Dimethoxyphenyl) cyclopropyl-carboxylic acid of 0.81g (93.5%).

3.44mmol1-(3,4-Dimethoxyphenyl) cyclopropyl-carboxylic acid, 3.5mmolN-methylmorpholine, and 3.5mmol isobutyl chlorocarbonate to be dissolved in anhydrous tetrahydro furan and to stir 15 minutes at-15 DEG C.Subsequently, add 3.5mmol3-(1H-imidazoles-1-base) alkyl-1-amine and make this mixture heat to 0 DEG C and stir 12 hours.Under reduced pressure remove desolventizing and remaining oil is heavily dissolved in chloroform.Subsequently, by organic layer NaHCO ₃saturated aqueous solution washs 2 times, with after through Na ₂sO ₄dry and except desolventizing.Use device (HarrisonResearchLtd.), utilizes silica gel plate and the CHCl of layer thickness 2mm ₃/ MeOH gradient is as elution system, purifying is carried out by centrifugal force chromatography, produce N-(3-(1H-imidazoles-1-base) propyl group)-1-(3, the 4-Dimethoxyphenyl) cyclopropane-methane amide of 0.671g (59.3%).

After 30mL heavily dissolves in Shui diox, add 1.43mmolLawesson reagent, and this mixture is heated to 90 DEG C and stirs 8 hours.By removal of solvent under reduced pressure, the resistates stayed is dissolved in 50mL methylene dichloride.Organic layer passes through NaHCO ₃saturated aqueous solution washs three times, washes three times subsequently with water, dry, filters and removes organic solvent subsequently.Use centrifugal force-chromatographic apparatus (HarrisonResearchLtd.), utilize silica gel plate and the CHCl of layer thickness 2mm ₃/ MeOH gradient as elution system, by chromatography by compound purifying.

Yield: 0.33g (46.2%); Fusing point: 127.0-127.5 DEG C

¹HNMRδ1.1-1.2(t，2H)，1.55-1.6(t，2H)，2.0-2.1(m，2H)，3.5-3.6(m，2H)，3.7-3.8(s，6H)，4.1-4.2(t，2H)，6.8-6.9(m，3H)，7.65(s，1H)，7.75(s，1H)，8.8(m，1H)，9.05(s，1H；MSm/z346.0(M+H)，278.2(M-C3H ₃N ₂·)，177.1(M-C6H ₈N ₃S·)

Example 133-135

1 eq of triethylamine and the mixture of 3,4-dimethoxyaniline in diox are added into the solution of the stirring of corresponding ω-bromo alkyl acid chloride in temperature 0 DEG C.This solution is heated to room temperature and stirs 2 hours.Evaporating solvent, and remaining oil is heavily dissolved in methylene dichloride.Organic layer washed with water, dry, filter and under reduced pressure solvent.Use centrifugal force-chromatographic apparatus (HarrisonResearchLtd.), utilize silica gel plate and the CHCl of layer thickness 2mm ₃/ MeOH gradient as elution system, by chromatography by compound purifying.

During imidazoles and sodium hydride are suspended in and by this mixture under inert conditions stirring at room temperature 3 hours.Add ω-bromo-N-(3,4-dimethoxy-phenylf) alkylamide and this mixture be heated to 100 DEG C and stir 8 hours, after this, evaporating solvent, adds hot toluene, and is filtered by solution.Subsequently, under elevated pressure except desolventizing.By Lawesson reagent, as described in example 113-132 is described, carry out the conversion of generation thioamides, produce 133-135 with total recovery.

As described below according to the analytical data of other example of above-mentioned general synthetic schemes synthesis:

Example 1:1-(3-(1H-imidazoles-1-base) propyl group)-3-methylthiourea

Fusing point: 122-122.5 DEG C

¹HNMRδ1.85-1.95(m，2H)，2.8(s，3H)，3.2-3.5(brd，2H)，3.8-3.9(m，2H)，6.85(d，1H)，7.15(d，1H)，7.3-7.5(brd，2H)，7.65(s，1H)；MSm/z199.1(M+H)，221.3(M+Na)，131.0(M-C3H ₃N ₂·)

Example 2:1-(3-(1H-imidazoles-1-base) propyl group)-3-tertiary butyl thiocarbamide

Fusing point: 147.0-147.5 DEG C

¹HNMRδ1.3-1.4(s，9H)，1.85-1.95(m，2H)，3.5(t，2H)，3.8(t，2H)，6.85(d，1H)，7.15(d，1H)，7.3-7.5(brd，2H)，7.65(s，1H)；MSm/z241.1(M+H)，173.1(M-C3H ₃N ₂·)

Example 3:1-(3-(1H-imidazoles-1-base) propyl group)-3-benzylthiourea

Fusing point: 127.0-128.0 DEG C

¹HNMRδ1.85-1.95(m，2H)，3.2-3.5(brd，2H)，3.8-3.9(m，2H)，4.6(s，2H)，6.8(d，1H)，7.15(d，1H)，7.19-7.35(m，5H)，7.5-7.6(brd，2H)，7.85(s，1H)；MSm/z275.3(M+H)，207.1(M-C3H ₃N ₂·)

Example 5:1-(3-(1H-imidazoles-1-base) propyl group)-3-phenylthiourea

Fusing point: 166.5-167.0 DEG C

¹HNMRδ1.95-2.05(m，2H)，3.3-3.5(brd，2H)，3.9-4.0(m，2H)，6.85(d，1H)，7.05(m，1H)7.15(d，1H)，7.25(m，2H)，7.35(m，2H)，7.6(s，1H)，7.8(brs，1H)，9.5(brs，1H)；MSm/z261.1(M+H)，193.2(M-C3H ₃N ₂·)

Example 6:1-(3-(1H-imidazoles-1-base) propyl group)-3-(4-fluorophenyl) thiocarbamide

Fusing point: 147.0-148.0 DEG C

¹HNMRδ1.95-2.05(m，2H)，3.3-3.5(brd，2H)，3.9-4.05(m，2H)，6.85(d，1H)，7.05-7.15(m，3H)，7.3-7.4(m，2H)，7.6(s，1H)，7.7-7.8(brs，1H)，9.4(brs，1H)；MSm/z279.3(M+H)，211.2(M-C3H ₃N ₂·)

Example 7:1-(3-(1H-imidazoles-1-base) propyl group)-3-(4-ethylphenyl) thiocarbamide

Fusing point: 100.0-100.5 DEG C

¹HNMRδ1.15-1.2(t，3H)，1.9-2.0(m，2H)，2.5-2.6(m，2H)，3.3-3.5(brd，2H)，3.9-4.05(m，2H)，6.85(d，1H)，7.1-7.2(m，3H)，7.25-7.3(m，2H)，7.6(s，1H)，7.7-7.8(brs，1H)，9.4(brs，1H)；MSm/z289.3(M+H)，221.1(M-C3H ₃N ₂·)

Example 8:1-(3-(1H-imidazoles-1-base) propyl group)-3-(4-(trifluoromethyl) phenyl) thiocarbamide

Fusing point: 154.5-155.0 DEG C

¹HNMRδ1.9-2.1(brm，2H)，3.4-3.6(brd，2H)，3.95-4.1(brm，2H)，6.85(d，1H)，7.2(d，1H)，7.6-7.8(m，5H)，8.2(brs，1H)，9.9(brs，1H)；MSm/z329.3(M+H)，261.2(M-C3H ₃N ₂·)

Example 10:1-(3-(1H-imidazoles-1-base) propyl group)-3-(4-acetyl phenyl) thiocarbamide

Fusing point: 170.0-171.0 DEG C

¹HNMRδ1.9-2.1(brm，2H)，2.4-2.5(s，3H)，3.2-3.5(brm，2H)，3.9-4.1(m，2H)，6.85(d，1H)，7.15(d，1H)，7.5-7.65(brm，3H)，7.8-7.9(m，2H)，8.1(m，2H)，9.8(brs，1H)；MSm/z303.2(M+H)，235.1(M-C3H ₃N ₂·)

Example 11:1-(3-(1H-imidazoles-1-base) propyl group)-3-(4-p-methoxy-phenyl) thiocarbamide

Fusing point: 125.0-125.5 DEG C

¹HNMRδ1.8-2.0(brm，2H)，3.2-3.5(brm，2H)，3.7(s，3H)，3.9-4.0(m，2H)，6.7-6.9(m，3H)，7.1-7.2(m，3H)，7.5(s，1H)，7.6(s，1H)，9.2(s，1H)；MSm/z291.1(M+H)，223.2(M-C3H ₃N ₂·)

Example 14:1-(3-(1H-imidazoles-1-base) propyl group)-3-(2,4-Dimethoxyphenyl) thiocarbamide

Fusing point: 120.0-120.5 DEG C

¹HNMRδ1.8-2.0(brm，2H)，3.4-3.5(brm，2H)，3.75(s，6H)，3.9-4.0(m，2H)，6.5(d，1H)，6.6(s，1H)，6.9(s，1H)，7.15(s，1H)，7.3(d，1H)，7.5(brs，1H)，7.6(s，1H)，9.75(s，1H)；MSm/z321.2(M+H)，253.3(M-C3H ₃N ₂·)

Example 15:1-(3-(1H-imidazoles-1-base) propyl group)-3-(3,5-Dimethoxyphenyl) thiocarbamide

Fusing point: 142.0-143.0 DEG C

¹HNMRδ1.8-2.0(brm，2H)，3.4-3.5(brm，2H)，3.6(s，6H)，3.95-4.0(m，2H)，6.25(m，1H)，6.6(m，2H)，6.9(s，1H)，7.2(s，1H)，7.6(s，1H)，7.8(s，1H)，9.5(s，1H)；MSm/z321.2(M+H)，253.3(M-C3H ₃N ₂·)

Example 23:1-(3-(1H-imidazoles-1-base) propyl group)-3-(2,3-dihydrobenzo [b] [Isosorbide-5-Nitrae] dioxin-7-base)-thiocarbamide

Fusing point: 103.0-103.5 DEG C

¹HNMRδ1.9-2.0(brm，2H)，3.3-3.5(brd，2H)，3.9-4.0(m，2H)，4.2-4.3(m，4H)，6.7(m，1H)，6.8-6.8(m，1H)，6.9(m，2H)，7.2(s，1H)，7.6(m，2H)，9.3(s，1H)；MSm/z319.3(M+H)，251.3(M-C3H ₃N ₂·)

Example 24:1-(3-(1H-imidazoles-1-base) propyl group)-3-(benzo [d] [1,3] dioxole-6-base) thiocarbamide

Fusing point: 115.6-115.6 DEG C

¹HNMRδ1.9-2.1(brm，2H)，3.4-3.5(brd，2H)，4.05-4.15(m，2H)，6.0(s，2H)，6.7(m，1H)，6.8-6.85(m，1H)，6.95(d，1H)，7.25(s，1H)，7.45(s，1H)，7.7(brs，1H)，8.5(brs，1H)，9.4(brs，1H)；MSm/z305.2(M+H)，237.2(M-C3H ₃N ₂·)

Example 25:1-(3-(1H-imidazoles-1-base) propyl group)-3-(3,4,5-trimethoxyphenyl) thiocarbamide

Fusing point: 124.5-125.5 DEG C

¹HNMRδ1.8-2.0(m，2H)，3.4-3.5(brm，2H)，3.6(s，3H)，3.7(s，6H)，3.9-4.0(m，2H)，6.65(m，2H)，6.85(s，1H)，7.2(s，1H)，7.6(s，1H)，7.7(brs，1H)，9.4(s，1H)；MSm/z351.3(M+H)，283.2(M-C3H ₃N ₂·)

Example 26:1-(3-(1H-imidazoles-1-base) propyl group)-3-(3-p-methoxy-phenyl) thiocarbamide

Fusing point: 89.5-90.0 DEG C

¹HNMRδ1.9-2.1(brm，2H)，3.4-3.5(brm，2H)，3.7(s，3H)，3.9-4.0(m，2H)，6.6-6.7(m，1H)，6.8-6.9(m，2H)，7.1(m，2H)，7.15-7.25(brm，1H)，7.6(s，1H)，7.8(brs，1H)，9.5(s，1H)；MSm/z291.1(M+H)，223.2(M-C3H ₃N ₂·)

Example 27:1-(3-(1H-imidazoles-1-base) propyl group)-3-(4-ethoxyl phenenyl) thiocarbamide

Fusing point: 126.0-126.5 DEG C

¹HNMRδ1.5(brm，3H)，1.9-2.0(brm，2H)，3.4-3.5(brm，2H)，3.9-4.0(brm，4H)，6.8-6.9(m，2H)，6.95(s，1H)，7.15-7.2(m，2H)，7.25(s，1H)，7.55-7.6(brs，1H)，7.8(s，1H)，9.3(s，1H)；MSm/z305.2(M+H)，237.2(M-C3H ₃N ₂·)

Example 33:1-(3-(1H-imidazoles-1-base) propyl group)-3-(4-(methylthio group) phenyl) thiocarbamide

Fusing point: 140.0-140.5 DEG C

¹HNMRδ1.8-2.05(brm，2H)，2.5(s，3H)，3.3-3.5(brm，2H)，3.9-4.1(m，2H)，6.9(m，1H)，7.1-7.3(brm，5H)，7.6(s，1H)，7.75(brs，1H)，9.4(s，1H)；MSm/z307.2(M+H)，239.2(M-C3H ₃N ₂·)

Example 42:1-(3-(1H-imidazoles-1-base) propyl group)-3-(4-nitrophenyl) thiocarbamide

Fusing point: 165.0.166.0 DEG C

¹HNMRδ1.9-2.05(m，2H)，3.3-3.5(brd，2H)，3.95-4.05(m，2H)，6.85(d，1H)，7.15(d，1H)，7.6(d，1H)，7.7(m，2H)，8.1(m，2H)，8.3(brs，1H)，10.1(brs，1H)；MSm/z306.2(M+H)，237.9(M-C3H ₃N ₂·)

Example 50:1-(3-(1H-imidazoles-1-base) propyl group)-3-(4-(dimethylamino) phenyl) thiocarbamide

Fusing point; 146.5-147.0 DEG C

¹HNMRδ1.9-2.0(m，2H)，2.9(s，6H)，3.4(m，2H)，3.9-4.0(m，2H)，6.7(m，2H)，6.9(s，1H)，7.05-7.1(m，2H)，7.15(s，1H)，7.4(brs，1H)，7.6(s，1H)，9.2(s，1H)；MSm/z304.2(M+H)，236.0(M-C3H ₃N ₂·)

Example 102:1-(3-(1H-imidazoles-1-base) propyl group)-3-(3,4-Dimethoxyphenyl) urea

Fusing point: 114.5-115.0 DEG C

¹HNMRδ1.7-1.9(m，2H)，2.9-3.1(m，2H)，3.7(2s，6H)，3.9-4.0(m，2H)，6.1(t，1H)，6.7(s，2H)，6.8(s，1H)，7.15(d，2H)，7.6(s，1H)，8.2(s，1H)；MSm/z321.2(M+H)，253.3(M-C3H ₃N ₂·)

Example 106:1-((S)-3-(1H-imidazoles-1-base)-2-methyl-propyl)-3-(3,4-Dimethoxyphenyl)-thiocarbamide

Fusing point:: 150.5-151.5 DEG C

¹HNMRδ0.9(d，3H)，2.3-2.4(m，2H)，2.5(s，1H)，3.7(d，6H)，4.0-4.1(brm，1H)，4.15-4.25(brm，1H)，6.75-6.8(m，1H)，6.85(m，1H)，6.9-7.0(m，1H)，7.65(s，1H)，7.75(s，2H)，9.1(s，1H)，9.5(s，1H)；MSm/z335.6(M+H)，267.1(M-C3H ₃N ₂·)

Example 107:1-((R)-3-(1H-imidazoles-1-base)-2-methyl-propyl)-3-(3,4-Dimethoxyphenyl)-thiocarbamide

Fusing point: 155.0-157.5 DEG C

¹HNMRδ0.9(d，3H)，2.3-2.4(m，2H)，2.5(s，1H)，3.7(d，6H)，4.0-4.1(brm，1H)，4.15-4.25(brm，1H)，6.75-6.8(m，1H)，6.85(m，1H)，6.9-7.0(m，1H)，7.65(s，1H)，7.75(s，2H)，9.1(s，1H)，9.5(s，1H)；MSm/z335.4(M+H)，267.2(M-C3H ₃N ₂·)

Example 109:1-((1-((1H-imidazoles-1-base) methyl) cyclopropyl) methyl)-3-(3,4-dimethoxy-phenylf) thiocarbamide

Fusing point: 166.5-168.5 DEG C

¹HNMRδ0.7-0.8(brm，2H)，1.85-1.9(m，1H)，2.15-2.2(m，1H)，2.2-2.3(m，1H)，3.4-3.5(m，1H)，3.7(d，6H)，4.2(s，1H)，4.95(s，1H)，6.75-6.8(brm，1H)，6.85-6.9(brm，1H)，7.0(s，1H)，7.5(m，1H)，7.6(m，1H)，7.7(s，0.5H)，7.8(s，0.5H)，8.85(s，0.5H)，9.1(s，0.5H)，9.35(s，0.5H)，9.45(s，0.5H)；MSm/z347.2(M+H)，279.2(M-C3H ₃N ₂·)，137.5(M-C ₉H ₁₃N ₄S·)

Example 110:N-(3-(1H-imidazoles-1-base) propyl group) benzo [d] thiazole-2-amine

¹HNMRδ1.95-2.15(m，2H)，3.25-3.35(m，2H)，4.0-4.1(t，2H)，6.9(s，1H)，6.95-7.05(t，1H)，7.15-7.2(m，2H)，7.35-7.4(d，1H)，7.60-7.70(m，2H)，8.0-8.1(brs，1H)；MSm/z259.4(M+H)，191.3(M-C3H ₃N ₂·)

Example 111:N-(3-(1H-imidazoles-1-base) propyl group)-6-chlorobenzene also [d] thiazole-2-amine

¹HNMRδ1.95-2.15(m，2H)，3.25-3.35(m，2H)，4.0-4.1(t，2H)，6.9(s，1H)，7.1-7.2(d，2H)，7.3-7.4(d，1H)，7.65(s，1H)，7.8(s，1H)，8.2(s，1H)；MSm/z293.3(M+H)，225.3(M-C3H ₃N ₂·)

Example 112:N-(3-(1H-imidazoles-1-base) propyl group)-6-methoxyl group benzo [d] thiazole-2-amine

¹HNMRδ1.9-2.05(m，2H)，3.2-3.3(m，2H)，3.7(s，3H)，4.0-4.1(t，2H)，6.7-6.8(d，1H)，6.9(s，1H)，7.15-7.2(s，1H)，7.2-7.3(m，2H)，7.65(s，1H)，7.8(s，1H)；MSm/z289.1(M+H)，221.4(M-C3H ₃N ₂·)

Example 115:(R)-N-(3-(1H-imidazoles-1-base) propyl group)-2-phenyl propionyl

Fusing point: 82.0-82.5 DEG C

¹HNMRδ1.4-1.55(d，3H)，1.9-2.0(m，2H)，3.4-3.5(m，2H)，3.85-3.95(m，2H)，4.0-4.1(q，1H)，6.8-6.9(s，1H)，7.1(s，1H)，7.15-7.2(m，1H)，7.2-7.3(m，2H)，7.35-7.4(m，2H)，7.55(s，1H)，10.1(s，1H)；MSm/z274.4(M+H)，206.3(M-C3H ₃N ₂·)

Example 116:(S)-N-(3-(1H-imidazoles-1-base) propyl group)-2-phenyl propionyl

Fusing point: 82.5-83.5 DEG C

Example 121:N-(3-(1H-imidazoles-1-base) propyl group)-1-(4-chloro-phenyl-) cyclobutylmethyl sulphamide

Fusing point: 137.5-139.0 DEG C

¹HNMRδ1.55-1.75(brm，2H)，1.85-1.95(brm，2H)，2.4-2.5(brm，2H)，2.7-2.85(brm，2H)，3.3-3.5(brm，2H)，3.8(m，2H)，6.9(s，1H)，7.0(s，1H)，7.3(m，2H)，7.45(s，1H)，7.5(m，2H)，9.6(t，1H)；MSm/z334.3(M+H)，266.1(M-C3H ₃N ₂·)

Example 122:N-(3-(1H-imidazoles-1-base) propyl group)-1-(4-chloro-phenyl-) cyclopentyl first sulphamide

Fusing point: 140.0-141.0 DEG C

¹HNMRδ1.5-1.65(brm，4H)，1.8-1.9(m，2H)，2.0-2.1(m，2H)，2.6(m，2H)，3.4-3.5(m，2H)，3.7-3.8(m，2H)，6.85(s，1H)，7.0(s，1H)，7.35(m，2H)，7.4(m，2H)，7.5(s，1H)，9.4(t，1H)；MSm/z348.2(M+H)，280.2(M-C3H ₃N ₂·)

Example 123:N-(3-(1H-imidazoles-1-base) propyl group)-1-(4-p-methoxy-phenyl) cyclohexyl first sulphamide

Fusing point: 162.5-164.0 DEG C

¹HNMRδ1.2-1.3(m，1H)，1.35-1.5(brm，5H)，1.85-2.0(brm，4H)，2.4-2.6(brm，2H)，3.4-3.5(m，2H)，3.7(s，3H)，3.8(m，2H)，6.8(m，3H)，7.0(s，1H)，7.3(m，2H)，7.5(s，1H)，9.2(t，1H)；MSm/z358.3(M+H)，2903(M-C3H ₃N ₂·)

Example 124:N-(3-(1H-imidazoles-1-base) propyl group)-1-(4-p-methoxy-phenyl) cyclopropyl first sulphamide

Fusing point:: 129.0-129.5 DEG C

¹HNMRδ1.0-1.1(m，2H)，1.5-1.6(m，2H)，1.9-2.0(brm，2H)，3.4-3.5(m，2H)，3.7(s，3H)，3.9(m，2H)，6.9(m，3H)，7.1(s，1H)，7.2-7.3(m，2H)，7.6(s，1H)，8.9(brs，1H)；MSm/z316.0(M+H)，248.4(M-C3H ₃N ₂·)

Example 134:5-(1H-imidazoles-1-base)-N-(3,4-Dimethoxyphenyl) amyl group sulphamide

Fusing point:: 128.0-128.5 DEG C

¹HNMRδ1.65-1.70(m，2H)，1.75-1.80(m，2H)，2.7-2.75(m，2H)，3.7(s，3H)，3.75(s，3H)，4.0-4.05(t，2H)，6.9-7.0(m，2H)，7.2(s，1H)，73(d，1H)，7.5(s，1H)，7.75(s，1H)，11.0(s，1H)；MSm/z320.2(M+H)，252.2(M-C3H ₃N ₂·)

Example 136:1-(2-(1H-imidazoles-1-base) ethyl)-3-(3,4-Dimethoxyphenyl) thiocarbamide

Fusing point: 157.5-159.0 DEG C

¹HNMRδ3.7(2s，6H)，3.8(m，2H)，4.2(m，2H)，6.7(m，1H)，6.85(m，1H)，6.9(m，2H)，7.15(s，1H)，7.5(brs，1H)，7.6(s，1H)，9.5(s，1H)；MSm/z307.2(M+H)，239.1(M-C3H ₃N ₂·)

Abbreviation

DEG C degree Celsius

A, Ala L-Ala

A amyloid beta-β peptide

4 amyloid in ABri Familial British Dementia

AC adenylyl cyclase

4 amyloid in ADan Familial Danish Dementia

AIM lacks in melanoma

AMC amino methylcoumarin

As antisense

Asp aspartic acid

β NA beta-naphthylamine

BA butyric acid

Bp base pair

BSA bovine serum albumin

C halfcystine

CAT E.C. 2.3.1.28

CAMP cAMP

CCL2MCP-1, MCP 1

CCL7MCP-3, monocyte chemotactic protein 3

CCL8MCP-2, monocyte chemotactic protein-2

CCL13MCP-4, monocyte chemoattractant protein-4

CDNA copy-DNA

C-His carboxyl terminal is histidine-tagged

CIDP chronic inflammatory demyelinating polyneuropathy

Cl chlorine

CSF celiolymph

C-terminus C-terminal

CTL cytotoxic T-lymphocytes

CV column volume

D diameter

Da dalton

DMSO dimethyl sulfoxide (DMSO)

DNA deoxyribonucleotide

E enzyme

EBVEpsteinBarr virus

ECL is ECL

E.coli intestinal bacteria

EC glutamyl cyclase

ED effective dose

EGFP enhanced green fluorescence protein

ES enzyme-substrate complex

FPP fertilization promotion peptide

FTC follicular thyroid carcinoma

G relative centrifugal force

GBSGuillain-Barr é syndrome

GF gel-filtration

Gln glutamine

Glu L-glutamic acid

GnRH gonadotropin releasing hormone (gonadoliberin)

GST glutathione S-transferase

H hydrogen

H people, hour

HGF pHGF

HIC hydrophobic interaction chromatography

HIF1a hypoxic inducing factor-1 a

His Histidine

HPLC high performance liquid chromatography

I inhibitor, Isoleucine

ID identity

IMAC immobilized metal affinity chromatography

IPTG isopropyl-beta D-thio galactopyranoside

K potassium

K constant

KDA kilodalton

K _iinhibitor constant

KLH keyhole (worm relative) hemocyanin

L length

LBLuria-Bertani

LD lethal dose

LPS lipopolysaccharides

M mole

μ l microlitre

μM micromole

MAGEA melanoma antigen family A

MAGEB melanoma antigen family B

Maldi-tof is substance assistant laser desorpted/the ionization flight time

MART1 is by the melanoma antigen 1 of T-cell recognition

Max maximum value

MCL-1 medullary cell leukemia 1

Met methionine(Met)

Min minute

MM mmole

MS multiple sclerosis

MRNA messenger RNA(mRNA)

N l-asparagine

Na sodium

NADH Reduced nicotinamide-adenine dinucleotide

Nm nanometer

NO numbers

NT neurotensin

Aminoterminal aminoterminal

O oxygen

OD optical density(OD)

P product, phosphorescent substance

PBS phosphate buffered saline (PBS)

PCR polymerase chain reaction

PGlu Pyrrolidonecarboxylic acid

PH acidity

Pro proline(Pro)

PTC papillary thyroid carcinoma

Pyr pyroglutamic acid ester

QC glutaminyl cyclase (glutamine peptide cyclotransferase)

The quantitative real-time polymerase chain reaction of qPCR

QPCTL glutamine peptide cyclotransferase sample

RNA ribonucleotide

RT reverse transcription; Reversed transcriptive enzyme

S substrate

S has justice

SAGE serial analysis of gene expression method

SDS sodium lauryl sulphate

SDS-PAGESDS-polyacrylamide gel electrophoresis

SGAP streptomyces griseus aminopeptidase

SEQ sequence

SNP single nucleotide polymorphism

Taa tumor associated antigen

TGF-β transforming growth factor-beta

TNF-cachectin α

TRH throtropin releasing hormone (thyroliberin)

TSH thyrotropin

TYR tyrosine oxidase

TYRP tyrosinase-related protein

U unit

UTC undifferentiated thyroid carcinoma

UV ultraviolet

V speed

VpAP Vibrio (Vibrio) proteolysis aminopeptidase

YSS yeast signal sequence

Zn zinc

Sequence table

<110> Probiodrug Gesellschaft Fur Arzneimittelforschung MBH

The new gene that <120> is relevant to glutaminyl cyclase

<130>PBD00060/WO

<150>US60/846,244

<151>2006-09-21

<150>US60/947,780

<151>2007-07-03

<160>121

<170>PatentInversion3.1

<210>1

<211>1086

<212>DNA

<213>human

<400>1

atggcaggcggaagacaccggcgcgtcgtgggcaccctccacctgctgctgctggtggcc60

gccctgccctgggcatccaggggggtcagtccgagtgcctcagcctggccagaggagaag120

aattaccaccagccagccattttgaattcatcggctcttcggcaaattgcagaaggcacc180

agtatctctgaaatgtggcaaaatgacttacagccattgctgatagagcgatacccggga240

tcccctggaagctatgctgctcgtcagcacatcatgcagcgaattcagaggcttcaggct300

gactgggtcttggaaatagacaccttcttgagtcagacaccctatgggtaccggtctttc360

tcaaatatcatcagcaccctcaatcccactgctaaacgacatttggtcctcgcctgccac420

tatgactccaagtatttttcccactggaacaacagagtgtttgtaggagccactgattca480

gccgtgccatgtgcaatgatgttggaacttgctcgtgccttagacaagaaactcctttcc540

ttaaagactgtttcagactccaagccagatttgtcactccagctgatcttctttgatggt600

gaagaggcttttcttcactggtctcctcaagattctctctatgggtctcgacacttagct660

gcaaagatggcatcgaccccgcacccacctggagcgagaggcaccagccaactgcatggc720

atggatttattggtcttattggatttgattggagctccaaacccaacgtttcccaatttt780

tttccaaactcagccaggtggttcgaaagacttcaagcaattgaacatgaacttcatgaa840

ttgggtttgctcaaggatcactctttggaggggcggtatttccagaattacagttatgga900

ggtgtgattcaggatgaccatattccatttttaagaagaggtgttccagttctgcatctg960

ataccgtctcctttccctgaagtctggcacaccatggatgacaatgaagaaaatttggat1020

gaatcaaccattgacaatctaaacaaaatcctacaagtctttgtgttggaatatcttcat1080

ttgtaa1086

<210>2

<211>1149

<212>DNA

<213>human

<400>2

atgcgttccgggggccgcgggcgaccccgcctgcggctgggggaacgtggcctcatggag60

ccactcttgccgccgaagcgccgcctgctaccgcgggttcggctcttgcctctgttgctg120

gcgctggccgtgggctcggcgttctacaccatttggagcggctggcaccgcaggactgag180

gagctgccgctgggccgggagctgcgggtcccattgatcggaagcctccccgaagcccgg240

ctgcggagggtggtgggacaactggatccacagcgtctctggagcacttatctgcgcccc300

ctgctggttgtgcgaaccccgggcagcccgggaaatctccaagtcagaaagttcctggag360

gccacgctgcggtccctgacagcaggttggcacgtggagctggatcccttcacagcctca420

acacccctggggccagtggactttggcaatgtggtggccacactggacccaagggctgcc480

cgtcacctcacccttgcctgccattatgactcgaagctcttcccacccggatcgaccccc540

tttgtaggggccacggattcggctgtgccctgtgccctgctgctggagctggcccaagca600

cttgacctggagctgagcagggccaaaaaacaggcagccccggtgaccctgcaactgctc660

ttcttggatggtgaagaggcgctgaaggagtggggacccaaggactccctttacggttcc720

cggcacctggcccagctcatggagtctatacctcacagccccggccccaccaggatccag780

gctattgagctctttatgcttcttgatctcctgggagcccccaatcccaccttctacagc840

cacttccctcgcacggtccgctggttccatcggctgaggagcattgagaagcgtctgcac900

cgtttgaacctgctgcagtctcatccccaggaagtgatgtacttccaacccggggagccc960

tttggctctgtggaagacgaccacatccccttcctccgcagaggggtacccgtgctccat1020

ctcatctccacgcccttccctgctgtctggcacacccctgcggacaccgaggtcaatctc1080

cacccacccacggtacacaacttgtgccgcattctcgctgtgttcctggctgaatacctg1140

gggctctag1149

<210>3

<211>1145

<212>DNA

<213>human

<400>3

atgcgttccgggggccgcgggcgaccccgcctgcggctgggggaacgtggatggagccac60

tcttgccgccgaagcgccgcctgctaccgcgggttcggctcttgcctctgttgctggcgc120

tggccgtgggctcggcgttctacaccatttggagcggctggcaccgcaggactgaggagc180

tgccgctgggccgggagctgcgggtcccattgatcggaagcctccccgaagcccggctgc240

ggagggtggtgggacaactggatccacagcgtctctggagcacttatctgcgccccctgc300

tggttgtgcgaaccccgggcagcccgggaaatctccaagtcagaaagttcctggaggcca360

cgctgcggtccctgacagcaggttggcacgtggagctggatcccttcacagcctcaacac420

ccctggggccagtggactttggcaatgtggtggccacactggacccaagggctgcccgtc480

acctcacccttgcctgccattatgactcgaagctcttcccacccggatcgaccccctttg540

taggggccacggattcggctgtgccctgtgccctgctgctggagctggcccaagcacttg600

acctggagctgagcagggccaaaaaacaggcagccccggtgaccctgcaactgctcttct660

tggatggtgaagaggcgctgaaggagtggggacccaaggactccctttacggttcccggc720

acctggcccagctcatggagtctatacctcacagccccggccccaccaggatccaggcta780

ttgagctctttatgcttcttgatctcctgggagcccccaatcccaccttctacagccact840

tccctcgcacggtccgctggttccatcggctgaggagcattgagaagcgtctgcaccgtt900

tgaacctgctgcagtctcatccccaggaagtgatgtacttccaacccggggagccctttg960

gctctgtggaagacgaccacatccccttcctccgcagaggggtacccgtgctccatctca1020

tctccacgcccttccctgctgtctggcacacccctgcggacaccgaggtcaatctccacc1080

cacccacggtacacaacttgtgccgcattctcgctgtgttcctggctgaatacctggggc1140

tctag1145

<210>4

<211>1149

<212>DNA

<213>Macacafascicularis

<400>4

atgcgttccgggggccgcgggcggccccgcctgcggctaggggaacgtggcgttatggag60

ccactcttgcccccgaagcgccgcctgctaccgcgggttcggctcttgcccctgttgctg120

gcgctggccgtgggctcggcgttctacaccatttggagcggctggcaccgcaggactgag180

gagctgccgctgggccgggagctgcgggtcccgttgatcggaagccttcccgaagcccgg240

ctgcggagggtggtgggacaactggacccacagcgtctctggggcacttatctgcgcccc300

ctgctggttgtgcgaaccccaggcagcccgggaaatctccaagtcagaaagttcctggag360

gccacgctgcggtccctgacagcaggttggcacgtggagctggatcccttcacagcctcg420

acgcccctggggccagtggactttggcaatgtggtggccacgctggacccgggggctgcc480

cgtcacctcacccttgcctgccattatgactcgaagctcttcccacccggatcgaccccg540

tttgtaggggccacggactcggctgtgccctgtgccctgctgctggagctggcccaggca600

cttgacctggagctgagcagggccaaagaacaggcagccccggtgaccctgcaactgctc660

ttcctggatggtgaagaggcgctgaaggagtggggacccaaggactccctttacggttcc720

cggcacctggcccagctcatggagtctatacctcatagccccggccccaccaggatccag780

gctattgagctctttatgcttcttgatctcctgggagcccccaatcccaccttctacagc840

cacttccctcgcacggtccgctggttccatcggctgagaagcattgagaagcgtctgcac900

cgtttgaacctgctgcagtctcatccccaggaagtgatgtacttccaacccggggagccc960

ttcggctctgtggaagacgaccacatccccttcctccgcagaggggtccccgtgctccat1020

ctcatctctacgcccttccctgctgtctggcacacccctgcggacacagaggccaatctc1080

cacccgcccacggtacacaacttaagccgcattctggccgtgttcctggctgaatacctg1140

gggctctag1149

<210>5

<211>1149

<212>DNA

<213>Macacamulatta

<400>5

atgcgttccgggggccgcgggcggccccgcctgcggctaggggaacgtggcgttatggag60

ccactcttgcccccgaagcgccgcctgctaccgcgggttcggctcttgcccctgttgctg120

gcgctggccgtgggctcggcgttctacaccatttggagcggctggcaccgcaggactgag180

gagctgccgctgggccgggagctgcgggtcccgttgatcggaagccttcccgaagcccgg240

ctgcggagggtggtgggacaactggacccacagcgtctctggggcacttatctgcgcccc300

ctgctggttgtgcgaaccccaggcagcccgggaaatctccaagtcagaaagttcctggag360

gccacgctgcggtccctgacagcaggttggcacgtggagctggatcccttcacagcctcg420

acgcccctgggcccagtggactttggcaatgtggtggccacgctggacccgggggctgcc480

cgtcacctcacccttgcctgccattatgactcgaagctcttcccacccggatcgaccccg540

tttgtaggggccacagactcggctgtgccctgtgccctgctgctggagctggcccaggca600

cttgacctggagctgagcagggccaaagaacaggcagccccggtgaccctgcaactgctc660

ttcctggatggtgaagaggcgctgaaggagtggggacccaaggactccctttacggttcc720

cggcacctggcccagctcatggagtctatacctcatagccccggccccaccaggatccag780

gctattgagctctttatgcttcttgatctcctgggagcccccaatcccaccttctacagc840

cacttccctcgcacggtccgctggttccatcggctgagaagcattgagaagcgtctgcac900

cgtttgaacctgctgcagtctcatccccaggaagtgatgtacttccaacccggggagccc960

tttggctctgtggaagacgaccacatccccttcctccgcagaggggtccccgtgctccat1020

ctcatctctacgcccttccctgctgtctggcacacccctgcggacacagaggccaatctc1080

cacccgcccacggtacacaacttaagccgcattctggccgtgttcctggctgaatacctg1140

gggctctag1149

<210>6

<211>1152

<212>DNA

<213>Canisfamiliaris

<400>6

atgccttccgggggccgcgggcggtcccggctacggctcggggaacgtggcctcttggag60

ccgccctccccgcccaagcgccgcctgctcccgcgggcgcacttcttgcctctgcttctg120

ctggccctggccctggcttcggcgacctacaccatctggagcggctggcaccaccagact180

gaggagctgccgcggggccgggagctgcggggccgcttgatcggaagcctctccgaagcc240

cggctgcggcgggtggtggggcaactggacccacaccgtctctggaacacttatctgcgc300

cccctgctggttgtgcggaccccgggcagccccggcaatctccaagtcagaaagttcctg360

gaggctacactacggaccttgacagcaggctggcatgtggaactggaccccttcacagcc420

ttgacacccctggggccactggactttggcaatgtggtggccacgctggacccaggggct480

gcccgtcacctcacccttgcctgccattatgactccaagctcttcgcatctgagtcggtt540

ccctttgtgggggcaacagattcggctgtaccttgcgccctgctgctggagctggctcag600

gccctcgacagggagttgagtagggccaaggagcaggaagccccggtgactctgcagctg660

ctctttttggatggtgaagaagcactgaaggagtggggacccacagactccctctatggc720

tcccggcacctggcccagctcatggagtctgcaccccacagcccgggccccaccaggatc780

caggctatcgagctcttcatgctccttgatctcctgggtgccccgaatccaaacttctac840

agtcacttccctcatacagcccgctggttccatcggctgaggagcatcgagaagcgcctt900

caccgcatgaacctgctgcagtctcatccccaggaagtgatgtacttccagcccggggag960

ccccctggttctgtggaagatgaccacatccccttcctccgccgaggggtccctgtgctc1020

cacctcatctccatgcccttcccctccgtctggcacacccccgatgactctgaggccaac1080

ctgcacccacccaccgtacacaatctgagccgcatcctcgccgtgttcctggccgaatat1140

ctggggctctag1152

<210>7

<211>1152

<212>DNA

<213>Rattusnorvegicus

<400>7

atgagtccggccagccgcgggcggtctcggcagcggctcggggatcgcggcctcatgaaa60

ccaccctcactttccaagcgccgtcttctgccgcgggtgcagctcctgcccctgctgctg120

ctggcgctggccctgggcttggctttttatatcgtctggaatagctggcaccctggggtt180

gaggaggtatcacggagccgggatctgcgggtcccgctgatcggaagcctttcagaagcc240

aagctgcggcttgtggtagggcagctggatccacagcgtctctggggaacttttctgcgt300

cccttgttgattgtacgacccccaggtagtcctggcaatctccaagtgagaaagttcctg360

gaggctacgttgcagtccctatcggcaggctggcacgtggaactggacccattcacagcc420

tcaacccccttggggccactggacttcgggaacgtggtggccacccttgacccaggagct480

gcccgtcacctcaccctcgcctgccattatgactctaagttcttccctcctgggttaccc540

ccctttgtgggggccacagattcagccgtgccctgtgccctgcttctggagttagtccag600

gcccttgatgtcatgctgagcagaatcaagcagcaggcagcaccagtgaccctgcagctg660

ctcttcttggacggggaggaggcactgaaggagtggggaccaaaggactccctctatggt720

tcccggcacctagctcagatcatggagtctataccgcacagccctggccccaccaggatc780

caggctattgagctctttgtccttcttgaccttctgggagcgcccagtccaatcttcttc840

agtcacttcccccgcacagcccgctggttccaacgactgcggagcatcgagaagcgcctt900

caccgtctgaacctactgcagtctcacccccaggaagtgatgtacttccaacccggggag960

ccccctggccctgtggaagatgaccacatccccttccttcgcagaggggtcccggtgctc1020

cacctcattgcgatgcccttccctgccgtgtggcacacacctgctgacactgaggctaac1080

ctccacccgcccacggtgcacaacctgagccgcatcctcgccgtgttcctggctgagtac1140

ctgggtctctag1152

<210>8

<211>1152

<212>DNA

<213>Musmusculus

<400>8

atgagtcccgggagccgcgggcggccccggcagcggctcgaggatcgtggcctcatgaaa60

ccaccctcactttccaagcgccgtcttctgccgcgagtgcagttcctgcccctgctgctg120

ctggcgctggctatgggcttggctttctatatcgtctggaacagctggcaccctggggtt180

gaggagatgtcacggagccgggatctgcgggtcccgctgatcggaagcctttcagaagcc240

aagctgcggctggtggtagggcagctggatccgcagcgtctctggggaactttcctgcgt300

cccttattgattgtgcgacccccgggtagttctggcaatctccaagtgagaaagttcctg360

gaggctacgttgcagtccctgtcggcaggctggcatgttgaactggacccattcacggcc420

tcaacccccttggggccactggacttcgggaacgtggtggccacacttgacccaggagct480

gcccgtcacctcaccctcgcctgccattatgactctaagttcttccctccggggttgccc540

ccctttgtgggggccacagattcagctgtgccctgtgccctgcttctggagttggtccag600

gcccttgatgccatgctgagcagaatcaagcagcaggcagcaccggtgaccctgcagctg660

cttttcttggatggggaggaggcactgaaggagtggggaccaaaggactccctctatggc720

tcccggcacctagctcagatcatggagtctataccacacagccctggccccaccaggatc780

caggctattgagctctttgtcctcctcgaccttctgggagcatccagtccgatcttcttc840

agtcacttccctcgcacagcccgctggttccagcgactgaggagcattgagaagcgcctt900

caccggctgaacctactgcagtctcacccccaggaagtgatgtacttccaacccggggag960

ccccccggccctgtggaagatgaccacatccccttccttcgcagaggggtcccggtgctc1020

cacctcattgccacgcccttccctgctgtgtggcacacacctgctgacaccgaggccaac1080

ctccacccacccactgtgcataacctgagccgcatccttgctgtgttcctggccgagtac1140

ctgggactctag1152

<210>9

<211>1152

<212>DNA

<213>Bostaurus

<400>9

atgccttccgggggccgcgggcggccccggctccaggtcggggaacgcagccttttggag60

cgaccctcaccgcccaagcgccgcctgataccgcgggcacagctgttgccccagctgctg120

ctggctctgacggtagcctcggtgttctataccatttggaggatctggcatagccagact180

gaagagctaccgctggggcgggagctgcggggccctttgatcggaagcctccccgaagct240

cgggtgcggagggtagtggggcaactggaccctcaccgtctctggaacactttcctgcgc300

cctctgctggttgtacggactccgggcagcccgggcaatctccaagtgagaaagttcctg360

gaggctacgctgcggacactttcagcaggctggcatatagaactcgactccttcactgcc420

tccacacccgtggggccattggacttcagcaatgtggtggccacgctggacccaggggct480

gcccgccaccttacccttgcctgccattatgactccaagctcttcccatctgactcagcc540

ccctttgtgggggccacggattcggcagtgccttgctccctgctactggagctggcccaa600

gcccttgaccaggagctgggcaaagccaaggagagggcagcgccaatgaccttgcagctg660

atcttcctggatggtgaagaggcactgaagcagtggggacccaaggactcgctttatggc720

tcccggcacctggcccagctcatggagtctacaccccacggcctgggctccaccaggatc780

caggctattgagctctttatgcttcttgatctcctgggagcccccaacccgaccttctac840

agtcacttccctcgcacggcccgctggttccatcggctcaggagcattgagaagcgcctg900

caccgtctgaacctcctgcagtctcatccttgggaagtgatgtacttccagaccggggag960

ccccccggctccgtggaagacgaccacatcccgttcctccgccgaggagttcccgtgctc1020

cacctcatcgccacacccttcccctctgtctggcacacgtccgatgactccgaggccaac1080

ctgcacccacccacggtacacaacctgagccgcatcctggccgtgttcctggctgagtac1140

ctggggctctag1152

<210>10

<211>361

<212>PRT

<213>human

<400>10

MetAlaGlyGlyArgHisArgArgValValGlyThrLeuHisLeuLeu

151015

LeuLeuValAlaAlaLeuProTrpAlaSerArgGlyValSerProSer

202530

AlaSerAlaTrpProGluGluLysAsnTyrHisGlnProAlaIleLeu

354045

AsnSerSerAlaLeuArgGlnIleAlaGluGlyThrSerIleSerGlu

505560

MetTrpGlnAsnAspLeuGlnProLeuLeuIleGluArgTyrProGly

65707580

SerProGlySerTyrAlaAlaArgGlnHisIleMetGlnArgIleGln

859095

ArgLeuGlnAlaAspTrpValLeuGluIleAspThrPheLeuSerGln

100105110

ThrProTyrGlyTyrArgSerPheSerAsnIleIleSerThrLeuAsn

115120125

ProThrAlaLysArgHisLeuValLeuAlaCysHisTyrAspSerLys

130135140

TyrPheSerHisTrpAsnAsnArgValPheValGlyAlaThrAspSer

145150155160

AlaValProCysAlaMetMetLeuGluLeuAlaArgAlaLeuAspLys

165170175

LysLeuLeuSerLeuLysThrValSerAspSerLysProAspLeuSer

180185190

LeuGlnLeuIlePhePheAspGlyGluGluAlaPheLeuHisTrpSer

195200205

ProGlnAspSerLeuTyrGlySerArgHisLeuAlaAlaLysMetAla

210215220

SerThrProHisProProGlyAlaArgGlyThrSerGlnLeuHisGly

225230235240

MetAspLeuLeuValLeuLeuAspLeuIleGlyAlaProAsnProThr

245250255

PheProAsnPhePheProAsnSerAlaArgTrpPheGluArgLeuGln

260265270

AlaIleGluHisGluLeuHisGluLeuGlyLeuLeuLysAspHisSer

275280285

LeuGluGlyArgTyrPheGlnAsnTyrSerTyrGlyGlyValIleGln

290295300

AspAspHisIleProPheLeuArgArgGlyValProValLeuHisLeu

305310315320

IleProSerProPheProGluValTrpHisThrMetAspAspAsnGlu

325330335

GluAsnLeuAspGluSerThrIleAspAsnLeuAsnLysIleLeuGln

340345350

ValPheValLeuGluTyrLeuHisLeu

355360

<210>11

<211>382

<212>PRT

<213>human

<400>11

MetArgSerGlyGlyArgGlyArgProArgLeuArgLeuGlyGluArg

151015

GlyLeuMetGluProLeuLeuProProLysArgArgLeuLeuProArg

202530

ValArgLeuLeuProLeuLeuLeuAlaLeuAlaValGlySerAlaPhe

354045

TyrThrIleTrpSerGlyTrpHisArgArgThrGluGluLeuProLeu

505560

GlyArgGluLeuArgValProLeuIleGlySerLeuProGluAlaArg

65707580

LeuArgArgValValGlyGlnLeuAspProGlnArgLeuTrpSerThr

859095

TyrLeuArgProLeuLeuValValArgThrProGlySerProGlyAsn

100105110

LeuGlnValArgLysPheLeuGluAlaThrLeuArgSerLeuThrAla

115120125

GlyTrpHisValGluLeuAspProPheThrAlaSerThrProLeuGly

130135140

ProValAspPheGlyAsnValValAlaThrLeuAspProArgAlaAla

145150155160

ArgHisLeuThrLeuAlaCysHisTyrAspSerLysLeuPheProPro

165170175

GlySerThrProPheValGlyAlaThrAspSerAlaValProCysAla

180185190

LeuLeuLeuGluLeuAlaGlnAlaLeuAspLeuGluLeuSerArgAla

195200205

LysLysGlnAlaAlaProValThrLeuGlnLeuLeuPheLeuAspGly

210215220

GluGluAlaLeuLysGluTrpGlyProLysAspSerLeuTyrGlySer

225230235240

ArgHisLeuAlaGlnLeuMetGluSerIleProHisSerProGlyPro

245250255

ThrArgIleGlnAlaIleGluLeuPheMetLeuLeuAspLeuLeuGly

260265270

AlaProAsnProThrPheTyrSerHisPheProArgThrValArgTrp

275280285

PheHisArgLeuArgSerIleGluLysArgLeuHisArgLeuAsnLeu

290295300

LeuGlnSerHisProGlnGluValMetTyrPheGlnProGlyGluPro

305310315320

PheGlySerValGluAspAspHisIleProPheLeuArgArgGlyVal

325330335

ProValLeuHisLeuIleSerThrProPheProAlaValTrpHisThr

340345350

ProAlaAspThrGluValAsnLeuHisProProThrValHisAsnLeu

355360365

CysArgIleLeuAlaValPheLeuAlaGluTyrLeuGlyLeu

370375380

<210>12

<211>364

<212>PRT

<213>human

<400>12

MetGluProLeuLeuProProLysArgArgLeuLeuProArgValArg

151015

LeuLeuProLeuLeuLeuAlaLeuAlaValGlySerAlaPheTyrThr

202530

IleTrpSerGlyTrpHisArgArgThrGluGluLeuProLeuGlyArg

354045

GluLeuArgValProLeuIleGlySerLeuProGluAlaArgLeuArg

505560

ArgValValGlyGlnLeuAspProGlnArgLeuTrpSerThrTyrLeu

65707580

ArgProLeuLeuValValArgThrProGlySerProGlyAsnLeuGln

859095

ValArgLysPheLeuGluAlaThrLeuArgSerLeuThrAlaGlyTrp

100105110

HisValGluLeuAspProPheThrAlaSerThrProLeuGlyProVal

115120125

AspPheGlyAsnValValAlaThrLeuAspProArgAlaAlaArgHis

130135140

LeuThrLeuAlaCysHisTyrAspSerLysLeuPheProProGlySer

145150155160

ThrProPheValGlyAlaThrAspSerAlaValProCysAlaLeuLeu

165170175

LeuGluLeuAlaGlnAlaLeuAspLeuGluLeuSerArgAlaLysLys

180185190

GlnAlaAlaProValThrLeuGlnLeuLeuPheLeuAspGlyGluGlu

195200205

AlaLeuLysGluTrpGlyProLysAspSerLeuTyrGlySerArgHis

210215220

LeuAlaGlnLeuMetGluSerIleProHisSerProGlyProThrArg

225230235240

IleGlnAlaIleGluLeuPheMetLeuLeuAspLeuLeuGlyAlaPro

245250255

AsnProThrPheTyrSerHisPheProArgThrValArgTrpPheHis

260265270

ArgLeuArgSerIleGluLysArgLeuHisArgLeuAsnLeuLeuGln

275280285

SerHisProGlnGluValMetTyrPheGlnProGlyGluProPheGly

290295300

SerValGluAspAspHisIleProPheLeuArgArgGlyValProVal

305310315320

LeuHisLeuIleSerThrProPheProAlaValTrpHisThrProAla

325330335

AspThrGluValAsnLeuHisProProThrValHisAsnLeuCysArg

340345350

IleLeuAlaValPheLeuAlaGluTyrLeuGlyLeu

355360

<210>13

<211>382

<212>PRT

<213>Macacafascicularis

<400>13

MetArgSerGlyGlyArgGlyArgProArgLeuArgLeuGlyGluArg

151015

GlyValMetGluProLeuLeuProProLysArgArgLeuLeuProArg

202530

ValArgLeuLeuProLeuLeuLeuAlaLeuAlaValGlySerAlaPhe

354045

TyrThrIleTrpSerGlyTrpHisArgArgThrGluGluLeuProLeu

505560

GlyArgGluLeuArgValProLeuIleGlySerLeuProGluAlaArg

65707580

LeuArgArgValValGlyGlnLeuAspProGlnArgLeuTrpGlyThr

859095

TyrLeuArgProLeuLeuValValArgThrProGlySerProGlyAsn

100105110

LeuGlnValArgLysPheLeuGluAlaThrLeuArgSerLeuThrAla

115120125

GlyTrpHisValGluLeuAspProPheThrAlaSerThrProLeuGly

130135140

ProValAspPheGlyAsnValValAlaThrLeuAspProGlyAlaAla

145150155160

ArgHisLeuThrLeuAlaCysHisTyrAspSerLysLeuPheProPro

165170175

GlySerThrProPheValGlyAlaThrAspSerAlaValProCysAla

180185190

LeuLeuLeuGluLeuAlaGlnAlaLeuAspLeuGluLeuSerArgAla

195200205

LysGluGlnAlaAlaProValThrLeuGlnLeuLeuPheLeuAspGly

210215220

GluGluAlaLeuLysGluTrpGlyProLysAspSerLeuTyrGlySer

225230235240

ArgHisLeuAlaGlnLeuMetGluSerIleProHisSerProGlyPro

245250255

ThrArgIleGlnAlaIleGluLeuPheMetLeuLeuAspLeuLeuGly

260265270

AlaProAsnProThrPheTyrSerHisPheProArgThrValArgTrp

275280285

PheHisArgLeuArgSerIleGluLysArgLeuHisArgLeuAsnLeu

290295300

LeuGlnSerHisProGlnGluValMetTyrPheGlnProGlyGluPro

305310315320

PheGlySerValGluAspAspHisIleProPheLeuArgArgGlyVal

325330335

ProValLeuHisLeuIleSerThrProPheProAlaValTrpHisThr

340345350

ProAlaAspThrGluAlaAsnLeuHisProProThrValHisAsnLeu

355360365

SerArgIleLeuAlaValPheLeuAlaGluTyrLeuGlyLeu

370375380

<210>14

<211>382

<212>PRT

<213>Macacamulatta

<400>14

MetArgSerGlyGlyArgGlyArgProArgLeuArgLeuGlyGluArg

151015

GlyValMetGluProLeuLeuProProLysArgArgLeuLeuProArg

202530

ValArgLeuLeuProLeuLeuLeuAlaLeuAlaValGlySerAlaPhe

354045

TyrThrIleTrpSerGlyTrpHisArgArgThrGluGluLeuProLeu

505560

GlyArgGluLeuArgValProLeuIleGlySerLeuProGluAlaArg

65707580

LeuArgArgValValGlyGlnLeuAspProGlnArgLeuTrpGlyThr

859095

TyrLeuArgProLeuLeuValValArgThrProGlySerProGlyAsn

100105110

LeuGlnValArgLysPheLeuGluAlaThrLeuArgSerLeuThrAla

115120125

GlyTrpHisValGluLeuAspProPheThrAlaSerThrProLeuGly

130135140

ProValAspPheGlyAsnValValAlaThrLeuAspProGlyAlaAla

145150155160

ArgHisLeuThrLeuAlaCysHisTyrAspSerLysLeuPheProPro

165170175

GlySerThrProPheValGlyAlaThrAspSerAlaValProCysAla

180185190

LeuLeuLeuGluLeuAlaGlnAlaLeuAspLeuGluLeuSerArgAla

195200205

LysGluGlnAlaAlaProValThrLeuGlnLeuLeuPheLeuAspGly

210215220

GluGluAlaLeuLysGluTrpGlyProLysAspSerLeuTyrGlySer

225230235240

ArgHisLeuAlaGlnLeuMetGluSerIleProHisSerProGlyPro

245250255

ThrArgIleGlnAlaIleGluLeuPheMetLeuLeuAspLeuLeuGly

260265270

AlaProAsnProThrPheTyrSerHisPheProArgThrValArgTrp

275280285

PheHisArgLeuArgSerIleGluLysArgLeuHisArgLeuAsnLeu

290295300

LeuGlnSerHisProGlnGluValMetTyrPheGlnProGlyGluPro

305310315320

PheGlySerValGluAspAspHisIleProPheLeuArgArgGlyVal

325330335

ProValLeuHisLeuIleSerThrProPheProAlaValTrpHisThr

340345350

ProAlaAspThrGluAlaAsnLeuHisProProThrValHisAsnLeu

355360365

SerArgIleLeuAlaValPheLeuAlaGluTyrLeuGlyLeu

370375380

<210>15

<211>383

<212>PRT

<213>Canisfamiliaris

<400>15

MetProSerGlyGlyArgGlyArgSerArgLeuArgLeuGlyGluArg

151015

GlyLeuLeuGluProProSerProProLysArgArgLeuLeuProArg

202530

AlaHisPheLeuProLeuLeuLeuLeuAlaLeuAlaLeuAlaSerAla

354045

ThrTyrThrIleTrpSerGlyTrpHisHisGlnThrGluGluLeuPro

505560

ArgGlyArgGluLeuArgGlyArgLeuIleGlySerLeuSerGluAla

65707580

ArgLeuArgArgValValGlyGlnLeuAspProHisArgLeuTrpAsn

859095

ThrTyrLeuArgProLeuLeuValValArgThrProGlySerProGly

100105110

AsnLeuGlnValArgLysPheLeuGluAlaThrLeuArgThrLeuThr

115120125

AlaGlyTrpHisValGluLeuAspProPheThrAlaLeuThrProLeu

130135140

GlyProLeuAspPheGlyAsnValValAlaThrLeuAspProGlyAla

145150155160

AlaArgHisLeuThrLeuAlaCysHisTyrAspSerLysLeuPheAla

165170175

SerGluSerValProPheValGlyAlaThrAspSerAlaValProCys

180185190

AlaLeuLeuLeuGluLeuAlaGlnAlaLeuAspArgGluLeuSerArg

195200205

AlaLysGluGlnGluAlaProValThrLeuGlnLeuLeuPheLeuAsp

210215220

GlyGluGluAlaLeuLysGluTrpGlyProThrAspSerLeuTyrGly

225230235240

SerArgHisLeuAlaGlnLeuMetGluSerAlaProHisSerProGly

245250255

ProThrArgIleGlnAlaIleGluLeuPheMetLeuLeuAspLeuLeu

260265270

GlyAlaProAsnProAsnPheTyrSerHisPheProHisThrAlaArg

275280285

TrpPheHisArgLeuArgSerIleGluLysArgLeuHisArgMetAsn

290295300

LeuLeuGlnSerHisProGlnGluValMetTyrPheGlnProGlyGlu

305310315320

ProProGlySerValGluAspAspHisIleProPheLeuArgArgGly

325330335

ValProValLeuHisLeuIleSerMetProPheProSerValTrpHis

340345350

ThrProAspAspSerGluAlaAsnLeuHisProProThrValHisAsn

355360365

LeuSerArgIleLeuAlaValPheLeuAlaGluTyrLeuGlyLeu

370375380

<210>16

<211>383

<212>PRT

<213>Rattusnorvegicus

<400>16

MetSerProAlaSerArgGlyArgSerArgGlnArgLeuGlyAspArg

151015

GlyLeuMetLysProProSerLeuSerLysArgArgLeuLeuProArg

202530

ValGlnLeuLeuProLeuLeuLeuLeuAlaLeuAlaLeuGlyLeuAla

354045

PheTyrIleValTrpAsnSerTrpHisProGlyValGluGluValSer

505560

ArgSerArgAspLeuArgValProLeuIleGlySerLeuSerGluAla

65707580

LysLeuArgLeuValValGlyGlnLeuAspProGlnArgLeuTrpGly

859095

ThrPheLeuArgProLeuLeuIleValArgProProGlySerProGly

100105110

AsnLeuGlnValArgLysPheLeuGluAlaThrLeuGlnSerLeuSer

115120125

AlaGlyTrpHisValGluLeuAspProPheThrAlaSerThrProLeu

130135140

GlyProLeuAspPheGlyAsnValValAlaThrLeuAspProGlyAla

145150155160

AlaArgHisLeuThrLeuAlaCysHisTyrAspSerLysPhePhePro

165170175

ProGlyLeuProProPheValGlyAlaThrAspSerAlaValProCys

180185190

AlaLeuLeuLeuGluLeuValGlnAlaLeuAspValMetLeuSerArg

195200205

IleLysGlnGlnAlaAlaProValThrLeuGlnLeuLeuPheLeuAsp

210215220

GlyGluGluAlaLeuLysGluTrpGlyProLysAspSerLeuTyrGly

225230235240

SerArgHisLeuAlaGlnIleMetGluSerIleProHisSerProGly

245250255

ProThrArgIleGlnAlaIleGluLeuPheValLeuLeuAspLeuLeu

260265270

GlyAlaProSerProIlePhePheSerHisPheProArgThrAlaArg

275280285

TrpPheGlnArgLeuArgSerIleGluLysArgLeuHisArgLeuAsn

290295300

LeuLeuGlnSerHisProGlnGluValMetTyrPheGlnProGlyGlu

305310315320

ProProGlyProValGluAspAspHisIleProPheLeuArgArgGly

325330335

ValProValLeuHisLeuIleAlaMetProPheProAlaValTrpHis

340345350

ThrProAlaAspThrGluAlaAsnLeuHisProProThrValHisAsn

355360365

LeuSerArgIleLeuAlaValPheLeuAlaGluTyrLeuGlyLeu

370375380

<210>17

<211>383

<212>PRT

<213>Musmusculus

<400>17

MetSerProGlySerArgGlyArgProArgGlnArgLeuGluAspArg

151015

GlyLeuMetLysProProSerLeuSerLysArgArgLeuLeuProArg

202530

ValGlnPheLeuProLeuLeuLeuLeuAlaLeuAlaMetGlyLeuAla

354045

PheTyrIleValTrpAsnSerTrpHisProGlyValGluGluMetSer

505560

ArgSerArgAspLeuArgValProLeuIleGlySerLeuSerGluAla

65707580

LysLeuArgLeuValValGlyGlnLeuAspProGlnArgLeuTrpGly

859095

ThrPheLeuArgProLeuLeuIleValArgProProGlySerSerGly

100105110

AsnLeuGlnValArgLysPheLeuGluAlaThrLeuGlnSerLeuSer

115120125

AlaGlyTrpHisValGluLeuAspProPheThrAlaSerThrProLeu

130135140

GlyProLeuAspPheGlyAsnValValAlaThrLeuAspProGlyAla

145150155160

AlaArgHisLeuThrLeuAlaCysHisTyrAspSerLysPhePhePro

165170175

ProGlyLeuProProPheValGlyAlaThrAspSerAlaValProCys

180185190

AlaLeuLeuLeuGluLeuValGlnAlaLeuAspAlaMetLeuSerArg

195200205

IleLysGlnGlnAlaAlaProValThrLeuGlnLeuLeuPheLeuAsp

210215220

GlyGluGluAlaLeuLysGluTrpGlyProLysAspSerLeuTyrGly

225230235240

SerArgHisLeuAlaGlnIleMetGluSerIleProHisSerProGly

245250255

ProThrArgIleGlnAlaIleGluLeuPheValLeuLeuAspLeuLeu

260265270

GlyAlaSerSerProIlePhePheSerHisPheProArgThrAlaArg

275280285

TrpPheGlnArgLeuArgSerIleGluLysArgLeuHisArgLeuAsn

290295300

LeuLeuGlnSerHisProGlnGluValMetTyrPheGlnProGlyGlu

305310315320

ProProGlyProValGluAspAspHisIleProPheLeuArgArgGly

325330335

ValProValLeuHisLeuIleAlaThrProPheProAlaValTrpHis

340345350

ThrProAlaAspThrGluAlaAsnLeuHisProProThrValHisAsn

355360365

LeuSerArgIleLeuAlaValPheLeuAlaGluTyrLeuGlyLeu

370375380

<210>18

<211>383

<212>PRT

<213>Bostaurus

<400>18

MetProSerGlyGlyArgGlyArgProArgLeuGlnValGlyGluArg

151015

SerLeuLeuGluArgProSerProProLysArgArgLeuIleProArg

202530

AlaGlnLeuLeuProGlnLeuLeuLeuAlaLeuThrValAlaSerVal

354045

PheTyrThrIleTrpArgIleTrpHisSerGlnThrGluGluLeuPro

505560

LeuGlyArgGluLeuArgGlyProLeuIleGlySerLeuProGluAla

65707580

ArgValArgArgValValGlyGlnLeuAspProHisArgLeuTrpAsn

859095

ThrPheLeuArgProLeuLeuValValArgThrProGlySerProGly

100105110

AsnLeuGlnValArgLysPheLeuGluAlaThrLeuArgThrLeuSer

115120125

AlaGlyTrpHisIleGluLeuAspSerPheThrAlaSerThrProVal

130135140

GlyProLeuAspPheSerAsnValValAlaThrLeuAspProGlyAla

145150155160

AlaArgHisLeuThrLeuAlaCysHisTyrAspSerLysLeuPhePro

165170175

SerAspSerAlaProPheValGlyAlaThrAspSerAlaValProCys

180185190

SerLeuLeuLeuGluLeuAlaGlnAlaLeuAspGlnGluLeuGlyLys

195200205

AlaLysGluArgAlaAlaProMetThrLeuGlnLeuIlePheLeuAsp

210215220

GlyGluGluAlaLeuLysGlnTrpGlyProLysAspSerLeuTyrGly

225230235240

SerArgHisLeuAlaGlnLeuMetGluSerThrProHisGlyLeuGly

245250255

SerThrArgIleGlnAlaIleGluLeuPheMetLeuLeuAspLeuLeu

260265270

GlyAlaProAsnProThrPheTyrSerHisPheProArgThrAlaArg

275280285

TrpPheHisArgLeuArgSerIleGluLysArgLeuHisArgLeuAsn

290295300

LeuLeuGlnSerHisProTrpGluValMetTyrPheGlnThrGlyGlu

305310315320

ProProGlySerValGluAspAspHisIleProPheLeuArgArgGly

325330335

ValProValLeuHisLeuIleAlaThrProPheProSerValTrpHis

340345350

ThrSerAspAspSerGluAlaAsnLeuHisProProThrValHisAsn

355360365

LeuSerArgIleLeuAlaValPheLeuAlaGluTyrLeuGlyLeu

370375380

<210>19

<211>1457

<212>DNA

<213>human

<400>19

gtctggtacaggtttcagggcaaagcggccatgcgttccgggggccgcgggcgaccccgc60

ctgcggctgggggaacgtggcctcatggagccactcttgccgccgaagcgccgcctgcta120

ccgcgggttcggctcttgcctctgttgctggcgctggccgtgggctcggcgttctacacc180

atttggagcggctggcaccgcaggactgaggagctgccgctgggccgggagctgcgggtc240

ccattgatcggaagcctccccgaagcccggctgcggagggtggtgggacaactggatcca300

cagcgtctctggagcacttatctgcgccccctgctggttgtgcgaaccccgggcagcccg360

ggaaatctccaagtcagaaagttcctggaggccacgctgcggtccctgacagcaggttgg420

cacgtggagctggatcccttcacagcctcaacacccctggggccagtggactttggcaat480

gtggtggccacactggacccaagggctgcccgtcacctcacccttgcctgccattatgac540

tcgaagctcttcccacccggatcgaccccctttgtaggggccacggattcggctgtgccc600

tgtgccctgctgctggagctggcccaagcacttgacctggagctgagcagggccaaaaaa660

caggcagccccggtgaccctgcaactgctcttcttggatggtgaagaggcgctgaaggag720

tggggacccaaggactccctttacggttcccggcacctggcccagctcatggagtctata780

cctcacagccccggccccaccaggatccaggctattgagctctttatgcttcttgatctc840

ctgggagcccccaatcccaccttctacagccacttccctcgcacggtccgctggttccat900

cggctgaggagcattgagaagcgtctgcaccgtttgaacctgctgcagtctcatccccag960

gaagtgatgtacttccaacccggggagccctttggctctgtggaagacgaccacatcccc1020

ttcctccgcagaggggtacccgtgctccatctcatctccacgcccttccctgctgtctgg1080

cacacccctgcggacaccgaggtcaatctccacccacccacggtacacaacttgtgccgc1140

attctcgctgtgttcctggctgaatacctggggctctagcgtgcttggccaatgactgtg1200

gagaggactgtgagagagaaggtcccagcgggggccagtgaagctcaggcaggatctgcc1260

tagggtgtgctggtttgtccttttcatacctttgtctcctaattgtgctacaattggaag1320

accttctttcttttgattgtctcaagctgccacccttcaaggacagggaagagaccactg1380

tgggatgacagccagaggaataagaacttgctccctccccagaggtaaacacttggtcca1440

aaggtttgcagggacca1457

<210>20

<211>1088

<212>DNA

<213>human

<400>20

agcggccatgcgttccgggggccgcgggcgaccccgcctgcggctgggggaacgtggcct60

catggagccactcttgccgccgaagcgccgcctgctaecgcgggttcggctcttgcctct120

gttgctggcgctggccgtgggctcggcgttctacaccatttggagcggctggcaccgcag180

gactgaggagctgccgctgggccgggagctgcgggtcccattgatcggaagcctccccga240

agcccggctgcggagggtggtgggacaactggatccacagcgtctctggagcacttatct300

gcgccccctgctggttgtgcgaaccccgggcagcccgggaaatctccaagtcagaaaggc360

agccccggtgaccctgcaactgctcttcttggatggtgaagaggcgctgaaggagtgggg420

acccaaggactccctttacggttcccggcacctggcccagctcatggagtctatacctca480

cagccccggccccaccaggatccaggctattgagctctttatgcttcttgatctcctggg540

agcccccaatcccaccttctacagccacttccctcgcacggtccgctggttccatcggct600

gaggagcattgagaagcgtctgcaccgtttgaacctgctgcagtctcatccccaggaagt660

gatgtacttccaacccggggagccctttggctctgtggaagacgaccacatccccttcct720

ccgcagaggggtacccgtgctccatctcatctccacgcccttccctgctgtctggcacac780

ccctgcggacaccgaggtcaatctccacccacccacggtacacaacttgtgccgcattct840

cgctgtgttcctggctgaatacctggggctctagcgtgcttggccaatgactgtggagag900

gactgtgagagagaaggtcccagcgggggccagtgaagctcaggcaggatctgcctaggg960

tgtgctggtttgtccttttcatacctttgtctcctaattgtgctacaattggaagacctt1020

ctttcttttgattgtctcaagctgccacccttcaaggacagggaagagaccactgtggga1080

tgacagcc1088

<210>21

<211>481

<212>PRT

<213>human

<400>21

ValTrpTyrArgPheGlnGlyLysAlaAlaMetArgSerGlyGlyArg

151015

GlyArgProArgLeuArgLeuGlyGluArgGlyLeuMetGluProLeu

202530

LeuProProLysArgArgLeuLeuProArgValArgLeuLeuProLeu

354045

LeuLeuAlaLeuAlaValGlySerAlaPheTyrThrIleTrpSerGly

505560

TrpHisArgArgThrGluGluLeuProLeuGlyArgGluLeuArgVal

65707580

ProLeuIleGlySerLeuProGluAlaArgLeuArgArgValValGly

859095

GlnLeuAspProGlnArgLeuTrpSerThrTyrLeuArgProLeuLeu

100105110

ValValArgThrProGlySerProGlyAsnLeuGlnValArgLysPhe

115120125

LeuGluAlaThrLeuArgSerLeuThrAlaGlyTrpHisValGluLeu

130135140

AspProPheThrAlaSerThrProLeuGlyProValAspPheGlyAsn

145150155160

ValValAlaThrLeuAspProArgAlaAlaArgHisLeuThrLeuAla

165170175

CysHisTyrAspSerLysLeuPheProProGlySerThrProPheVal

180185190

GlyAlaThrAspSerAlaValProCysAlaLeuLeuLeuGluLeuAla

195200205

GlnAlaLeuAspLeuGluLeuSerArgAlaLysLysGlnAlaAlaPro

210215220

ValThrLeuGlnLeuLeuPheLeuAspGlyGluGluAlaLeuLysGlu

225230235240

TrpGlyProLysAspSerLeuTyrGlySerArgHisLeuAlaGlnLeu

245250255

MetGluSerIleProHisSerProGlyProThrArgIleGlnAlaIle

260265270

GluLeuPheMetLeuLeuAspLeuLeuGlyAlaProAsnProThrPhe

275280285

TyrSerHisPheProArgThrValArgTrpPheHisArgLeuArgSer

290295300

IleGluLysArgLeuHisArgLeuAsnLeuLeuGlnSerHisProGln

305310315320

GluValMetTyrPheGlnProGlyGluProPheGlySerValGluAsp

325330335

AspHisIleProPheLeuArgArgGlyValProValLeuHisLeuIle

340345350

SerThrProPheProAlaValTrpHisThrProAlaAspThrGluVal

355360365

AsnLeuHisProProThrValHisAsnLeuCysArgIleLeuAlaVal

370375380

PheLeuAlaGluTyrLeuGlyLeuArgAlaTrpProMetThrValGlu

385390395400

ArgThrValArgGluLysValProAlaGlyAlaSerGluAlaGlnAla

405410415

GlySerAlaGlyValLeuValCysProPheHisThrPheValSerLeu

420425430

CysTyrAsnTrpLysThrPhePheLeuLeuIleValSerSerCysHis

435440445

ProSerArgThrGlyLysArgProLeuTrpAspAspSerGlnArgAsn

450455460

LysAsnLeuLeuProProGlnArgThrLeuGlyProLysValCysArg

465470475480

Asp

<210>22

<211>359

<212>PRT

<213>human

<400>22

AlaAlaMetArgSerGlyGlyArgGlyArgProArgLeuArgLeuGly

151015

GluArgGlyLeuMetGluProLeuLeuProProLysArgArgLeuLeu

202530

ProArgValArgLeuLeuProLeuLeuLeuAlaLeuAlaValGlySer

354045

AlaPheTyrThrIleTrpSerGlyTrpHisArgArgThrGluGluLeu

505560

ProLeuGlyArgGluLeuArgValProLeuIleGlySerLeuProGlu

65707580

AlaArgLeuArgArgValValGlyGlnLeuAspProGlnArgLeuTrp

859095

SerThrTyrLeuArgProLeuLeuValValArgThrProGlySerPro

100105110

GlyAsnLeuGlnValArgLysAlaAlaProValThrLeuGlnLeuLeu

115120125

PheLeuAspGlyGluGluAlaLeuLysGluTrpGlyProLysAspSer

130135140

LeuTyrGlySerArgHisLeuAlaGlnLeuMetGluSerIleProHis

145150155160

SerProGlyProThrArgIleGlnAlaIleGluLeuPheMetLeuLeu

165170175

AspLeuLeuGlyAlaProAsnProThrPheTyrSerHisPheProArg

180185190

ThrValArgTrpPheHisArgLeuArgSerIleGluLysArgLeuHis

195200205

ArgLeuAsnLeuLeuGlnSerHisProGlnGluValMetTyrPheGln

210215220

ProGlyGluProPheGlySerValGluAspAspHisIleProPheLeu

225230235240

ArgArgGlyValProValLeuHisLeuIleSerThrProPheProAla

245250255

ValTrpHisThrProAlaAspThrGluValAsnLeuHisProProThr

260265270

ValHisAsnLeuCysArgIleLeuAlaValPheLeuAlaGluTyrLeu

275280285

GlyLeuArgAlaTrpProMetThrValGluArgThrValArgGluLys

290295300

ValProAlaGlyAlaSerGluAlaGlnAlaGlySerAlaGlyValLeu

305310315320

ValCysProPheHisThrPheValSerLeuCysTyrAsnTrpLysThr

325330335

PhePheLeuLeuIleValSerSerCysHisProSerArgThrGlyLys

340345350

ArgProLeuTrpAspAspSer

355

<210>23

<211>42

<212>PRT

<213>Homosapiens

<400>23

AspAlaGluPheArgHisAspSerGlyTyrGluValHisHisGlnLys

151015

LeuValPhePheAlaGluAspValGlySerAsnLysGlyAlaIleIle

202530

GlyLeuMetValGlyGlyValValIleAla

3540

<210>24

<211>40

<212>PRT

<213>Homosapiens

<400>24

AspAlaGluPheArgHisAspSerGlyTyrGluValHisHisGlnLys

151015

LeuValPhePheAlaGluAspValGlySerAsnLysGlyAlaIleIle

202530

GlyLeuMetValGlyGlyValVal

3540

<210>25

<211>40

<212>PRT

<213>Homosapiens

<400>25

GluPheArgHisAspSerGlyTyrGluValHisHisGlnLysLeuVal

151015

PhePheAlaGluAspValGlySerAsnLysGlyAlaIleIleGlyLeu

202530

MetValGlyGlyValValIleAla

3540

<210>26

<211>38

<212>PRT

<213>Homosapiens

<400>26

GluPheArgHisAspSerGlyTyrGluValHisHisGlnLysLeuVal

151015

PhePheAlaGluAspValGlySerAsnLysGlyAlaIleIleGlyLeu

202530

MetValGlyGlyValVal

35

<210>27

<211>32

<212>PRT

<213>artificialsequence

<220>

<223>syntheticpeptide

<400>27

GluValHisHisGlnLysLeuValPhePheAlaGluAspValGlySer

151015

AsnLysGlyAlaIleIleGlyLeuMetValGlyGlyValValIleAla

202530

<210>28

<211>30

<212>PRT

<213>artificialsequence

<220>

<223>syntheticpeptide

<400>28

GluValHisHisGlnLysLeuValPhePheAlaGluAspValGlySer

151015

AsnLysGlyAlaIleIleGlyLeuMetValGlyGlyValVal

202530

<210>29

<211>40

<212>PRT

<213>human

<220>

<221>MOD_RES

<222>(1)..(1)

<223>PYRROLIDONECARBOXYLICACID

<400>29

GluPheArgHisAspSerGlyTyrGluValHisHisGlnLysLeuVal

151015

PhePheAlaGluAspValGlySerAsnLysGlyAlaIleIleGlyLeu

202530

MetValGlyGlyValValIleAla

3540

<210>30

<211>38

<212>PRT

<213>human

<220>

<221>MOD_RES

<222>(1)..(1)

<223>PYRROLIDONECARBOXYLICACID

<400>30

GluPheArgHisAspSerGlyTyrGluValHisHisGlnLysLeuVal

151015

PhePheAlaGluAspValGlySerAsnLysGlyAlaIleIleGlyLeu

202530

MetValGlyGlyValVal

35

<210>31

<211>32

<212>PRT

<213>human

<220>

<221>MOD_RES

<222>(1)..(1)

<223>PYRROLIDONECARBOXYLICACID

<400>31

GluValHisHisGlnLysLeuValPhePheAlaGluAspValGlySer

151015

AsnLysGlyAlaIleIleGlyLeuMetValGlyGlyValValIleAla

202530

<210>32

<211>30

<212>PRT

<213>human

<220>

<221>MOD_RES

<222>(1)..(1)

<223>PYRROLIDONECARBOXYLICACID

<400>32

GluValHisHisGlnLysLeuValPhePheAlaGluAspValGlySer

151015

AsnLysGlyAlaIleIleGlyLeuMetValGlyGlyValVal

202530

<210>33

<211>34

<212>PRT

<213>Homosapiens

<400>33

GluAlaSerAsnCysPheAlaIleArgHisPheGluAsnLysPheAla

151015

ValGluThrLeuIleCysSerArgThrValLysLysAsnIleIleGlu

202530

GluArg

<210>34

<211>34

<212>PRT

<213>Homosapiens

<400>34

GluAlaSerAsnCysPheAlaIleArgHisPheGluAsnLysPheAla

151015

ValGluThrLeuIleCysSerArgThrValLysLysAsnIleIleGlu

202530

GluArg

<210>35

<211>17

<212>PRT

<213>Homosapiens

<220>

<221>MOD_RES

<222>(17)..(17)

<223>AMIDATION

<400>35

GlnGlyProTrpLeuGluGluGluGluGluAlaTyrGlyTrpMetAsp

151015

Phe

<210>36

<211>34

<212>PRT

<213>human

<400>36

GlnLeuGlyProGlnGlyProProHisLeuValAlaAspProSerLys

151015

LysGlnGlyProTrpLeuGluGluGluGluGluAlaTyrGlyTrpMet

202530

AspPhe

<210>37

<211>34

<212>PRT

<213>Homosapiens

<220>

<221>MOD_RES

<222>(1)..(1)

<223>PYRROLIDONECARBOXYLICACID

<400>37

GluAlaSerAsnCysPheAlaIleArgHisPheGluAsnLysPheAla

151015

ValGluThrLeuIleCysSerArgThrValLysLysAsnIleIleGlu

202530

GluArg

<210>38

<211>34

<212>PRT

<213>Homosapiens

<220>

<221>MOD_RES

<222>(1)..(1)

<223>PYRROLIDONECARBOXYLICACID

<400>38

GluAlaSerAsnCysPheAlaIleArgHisPheGluAsnLysPheAla

151015

ValGluThrLeuIleCysSerArgThrValLysLysAsnIleIleGlu

202530

GluArg

<210>39

<211>17

<212>PRT

<213>human

<220>

<221>MOD_RES

<222>(1)..(1)

<223>PYRROLIDONECARBOXYLICACID

<400>39

GlnGlyProTrpLeuGluGluGluGluGluAlaTyrGlyTrpMetAsp

151015

Phe

<210>40

<211>34

<212>PRT

<213>human

<220>

<221>MOD_RES

<222>(1)..(1)

<223>PYRROLIDONECARBOXYLICACID

<400>40

GlnLeuGlyProGlnGlyProProHisLeuValAlaAspProSerLys

151015

LysGlnGlyProTrpLeuGluGluGluGluGluAlaTyrGlyTrpMet

202530

AspPhe

<210>41

<211>13

<212>PRT

<213>Homosapiens

<400>41

GlnLeuTyrGluAsnLysProArgArgProTyrIleLeu

1510

<210>42

<211>10

<212>PRT

<213>Homosapiens

<220>

<221>MOD_RES

<222>(10)..(10)

<223>AMIDATION

<400>42

GlnHisTrpSerTyrGlyLeuArgProGly

1510

<210>43

<211>97

<212>PRT

<213>Homosapiens

<400>43

GlnProLysValProGluTrpValAsnThrProSerThrCysCysLeu

151015

LysTyrTyrGluLysValLeuProArgArgLeuValValGlyTyrArg

202530

LysAlaLeuAsnCysHisLeuProAlaIleIlePheValThrLysArg

354045

AsnArgGluValCysThrAsnProAsnAspAspTrpValGlnGluTyr

505560

IleLysAspProAsnLeuProLeuLeuProThrArgAsnLeuSerThr

65707580

ValLysIleIleThrAlaLysAsnGlyGlnProGlnLeuLeuAsnSer

859095

Gln

<210>44

<211>76

<212>PRT

<213>Homosapiens

<400>44

GlnProAspSerValSerIleProIleThrCysCysPheAsnValIle

151015

AsnArgLysIleProIleGlnArgLeuGluSerTyrThrArgIleThr

202530

AsnIleGlnCysProLysGluAlaValIlePheLysThrLysArgGly

354045

LysGluValCysAlaAspProLysGluArgTrpValArgAspSerMet

505560

LysHisLeuAspGlnIlePheGlnAsnLeuLysPro

657075

<210>45

<211>76

<212>PRT

<213>Homosapiens

<400>45

GlnProAspAlaIleAsnAlaProValThrCysCysTyrAsnPheThr

151015

AsnArgLysIleSerValGlnArgLeuAlaSerTyrArgArgIleThr

202530

SerSerLysCysProLysGluAlaValIlePheLysThrIleValAla

354045

LysGluIleCysAlaAspProLysGlnLysTrpValGlnAspSerMet

505560

AspHisLeuAspLysGlnThrGlnThrProLysThr

657075

<210>46

<211>68

<212>PRT

<213>Homosapiens

<400>46

GlnValGlyThrAsnLysGluLeuCysCysLeuValTyrThrSerTrp

151015

GlnIleProGlnLysPheIleValAspTyrSerGluThrSerProGln

202530

CysProLysProGlyValIleLeuLeuThrLysArgGlyArgGlnIle

354045

CysAlaAspProAsnLysLysTrpValGlnLysTyrIleSerAspLeu

505560

LysLeuAsnAla

65

<210>47

<211>373

<212>PRT

<213>Homosapiens

<400>47

GlnHisHisGlyValThrLysCysAsnIleThrCysSerLysMetThr

151015

SerLysIleProValAlaLeuLeuIleHisTyrGlnGlnAsnGlnAla

202530

SerCysGlyLysArgAlaIleIleLeuGluThrArgGlnHisArgLeu

354045

PheCysAlaAspProLysGluGlnTrpValLysAspAlaMetGlnHis

505560

LeuAspArgGlnAlaAlaAlaLeuThrArgAsnGlyGlyThrPheGlu

65707580

LysGlnIleGlyGluValLysProArgThrThrProAlaAlaGlyGly

859095

MetAspGluSerValValLeuGluProGluAlaThrGlyGluSerSer

100105110

SerLeuGluProThrProSerSerGlnGluAlaGlnArgAlaLeuGly

115120125

ThrSerProGluLeuProThrGlyValThrGlySerSerGlyThrArg

130135140

LeuProProThrProLysAlaGlnAspGlyGlyProValGlyThrGlu

145150155160

LeuPheArgValProProValSerThrAlaAlaThrTrpGlnSerSer

165170175

AlaProHisGlnProGlyProSerLeuTrpAlaGluAlaLysThrSer

180185190

GluAlaProSerThrGlnAspProSerThrGlnAlaSerThrAlaSer

195200205

SerProAlaProGluGluAsnAlaProSerGluGlyGlnArgValTrp

210215220

GlyGlnGlyGlnSerProArgProGluAsnSerLeuGluArgGluGlu

225230235240

MetGlyProValProAlaHisThrAspAlaPheGlnAspTrpGlyPro

245250255

GlySerMetAlaHisValSerValValProValSerSerGluGlyThr

260265270

ProSerArgGluProValAlaSerGlySerTrpThrProLysAlaGlu

275280285

GluProIleHisAlaThrMetAspProGlnArgLeuGlyValLeuIle

290295300

ThrProValProAspAlaGlnAlaAlaThrArgArgGlnAlaValGly

305310315320

LeuLeuAlaPheLeuGlyLeuLeuPheCysLeuGlyValAlaMetPhe

325330335

ThrTyrGlnSerLeuGlnGlyCysProArgLysMetAlaGlyGluMet

340345350

AlaGluGlyLeuArgTyrIleProArgSerCysGlySerAsnSerTyr

355360365

ValLeuValProVal

370

<210>48

<211>76

<212>PRT

<213>Homosapiens

<400>48

GlnProValGlyIleAsnThrSerThrThrCysCysTyrArgPheIle

151015

AsnLysLysIleProLysGlnArgLeuGluSerTyrArgArgThrThr

202530

SerSerHisCysProArgGluAlaValIlePheLysThrLysLeuAsp

354045

LysGluIleCysAlaAspProThrGlnLysTrpValGlnAspPheMet

505560

LysHisLeuAspLysLysThrGlnThrProLysLeu

657075

<210>49

<211>33

<212>PRT

<213>Homosapiens

<400>49

GlnProLeuProAspCysCysArgGlnLysThrCysSerCysArgLeu

151015

TyrGluLeuLeuHisGlyAlaGlyAsnHisAlaAlaGlyIleLeuThr

202530

Leu

<210>50

<211>11

<212>PRT

<213>Homosapiens

<400>50

ArgProLysProGlnGlnPhePheGlyLeuMet

1510

<210>51

<211>5

<212>PRT

<213>artificialsequence

<220>

<223>yntheticpeptide

<400>51

GlnTyrAsnAlaAsp

15

<210>52

<211>5

<212>PRT

<213>artificialsequence

<220>

<223>syntheticpeptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223>PYRROLIDONECARBOXYLICACID

<400>52

GlnTyrAsnAlaAsp

15

<210>53

<211>26

<212>DNA

<213>artificialsequence

<220>

<223>syntheticnucleotide

<400>53

ggtctacaccatttggagcggctggc26

<210>54

<211>27

<212>DNA

<213>artificialsequence

<220>

<223>synthticnucleotide

<400>54

gggttggaagtacatcacttcctgggg27

<210>55

<211>24

<212>DNA

<213>artificialsequence

<220>

<223>syntheticnucleotide

<400>55

accatgcgttccgggggccgcggg24

<210>56

<211>27

<212>DNA

<213>artificialsequence

<220>

<223>syntheticnucleotide

<400>56

acgctagagccccaggtattcagccag27

<210>57

<211>30

<212>DNA

<213>artificialsequence

<220>

<223>syntheticnucleotide

<400>57

atatatgaattcatgcgttccgggggccgc30

<210>58

<211>33

<212>DNA

<213>artificialsequence

<220>

<223>syntheticnucleotide

<400>58

atatatgaattcatggagccactcttgccgccg33

<210>59

<211>33

<212>DNA

<213>artificialsequence

<220>

<223>syntheticnucleotide

<400>59

atatatgtcgacgagccccaggtattcagccag33

<210>60

<211>44

<212>DNA

<213>artificialsequence

<220>

<223>syntheticnucleotide

<400>60

atatactagtgatgacgacgacaagttctacaccatttggagcg44

<210>61

<211>49

<212>DNA

<213>artificialsequence

<220>

<223>syntheticnucleotide

<400>61

tatagaattcctagtgatggtgatggtgatggagccccaggtattcagc49

<210>62

<211>28

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>62

atatgaattcttctacaccatttggagc28

<210>63

<211>49

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>63

atatgaattccatcaccatcaccatcacttctacaccatttggagcggc49

<210>64

<211>35

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>64

atatatgcggccgcctagagccccaggtattcagc35

<210>65

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>65

ccaggatccaggctattgag20

<210>66

<211>56

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>66

atatatgcggccgcctagtgatggtgatggtgatggagccccaggtattcagccag56

<210>67

<211>19

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>67

ttccacagggccggggggc19

<210>68

<211>18

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>68

atgagtcccgggagccgc18

<210>69

<211>18

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>69

ctagagtcccaggtactc18

<210>70

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>70

agttcctgcccctgctgctg20

<210>71

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>71

atcaagaggcaccaaccaac20

<210>72

<211>19

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>72

ctggataatatttccatag19

<210>73

<211>19

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>73

acagctgggaatctgagtc19

<210>74

<211>21

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>74

gagcagaatagcttccgggcg21

<210>75

<211>33

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>75

ctgcgggtcccattgaacggaagcctccccgaa33

<210>76

<211>33

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>76

ttcggggaggcttccgttcaatgggacccgcag33

<210>77

<211>33

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>77

acggtacacaacttggcccgcattctcgctgtg33

<210>78

<211>33

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>78

cacagcgagaatgcgggccaagttgtgtaccgt33

<210>79

<211>362

<212>PRT

<213>Musmusculus

<400>79

MetAlaGlySerGluAspLysLeuValValGlyThrLeuHisLeuLeu

151015

LeuLeuGlnAlaThrValLeuSerLeuThrAlaGlyAsnLeuSerLeu

202530

ValSerAlaAlaTrpThrGlnGluLysAsnHisHisGlnProAlaHis

354045

LeuAsnSerSerSerLeuGlnGlnValAlaGluGlyThrSerIleSer

505560

GluMetTrpGlnAsnAspLeuArgProLeuLeuIleGluArgTyrPro

65707580

GlySerProGlySerTyrSerAlaArgGlnHisIleMetGlnArgIle

859095

GlnArgLeuGlnAlaGluTrpValValGluValAspThrPheLeuSer

100105110

ArgThrProTyrGlyTyrArgSerPheSerAsnIleIleSerThrLeu

115120125

AsnProGluAlaLysArgHisLeuValLeuAlaCysHisTyrAspSer

130135140

LysTyrPheProArgTrpAspSerArgValPheValGlyAlaThrAsp

145150155160

SerAlaValProCysAlaMetMetLeuGluLeuAlaArgAlaLeuAsp

165170175

LysLysLeuHisSerLeuLysAspValSerGlySerLysProAspLeu

180185190

SerLeuArgLeuIlePhePheAspGlyGluGluAlaPheHisHisTrp

195200205

SerProGlnAspSerLeuTyrGlySerArgHisLeuAlaGlnLysMet

210215220

AlaSerSerProHisProProGlySerArgGlyThrAsnGlnLeuAsp

225230235240

GlyMetAspLeuLeuValLeuLeuAspLeuIleGlyAlaAlaAsnPro

245250255

ThrPheProAsnPhePheProLysThrThrArgTrpPheAsnArgLeu

260265270

GlnAlaIleGluLysGluLeuTyrGluLeuGlyLeuLeuLysAspHis

275280285

SerLeuGluArgLysTyrPheGlnAsnPheGlyTyrGlyAsnIleIle

290295300

GlnAspAspHisIleProPheLeuArgLysGlyValProValLeuHis

305310315320

LeuIleAlaSerProPheProGluValTrpHisThrMetAspAspAsn

325330335

GluGluAsnLeuHisAlaSerThrIleAspAsnLeuAsnLysIleIle

340345350

GlnValPheValLeuGluTyrLeuHisLeu

355360

<210>80

<211>284

<212>PRT

<213>Strepromycesgriseus

<400>80

AlaProAspIleProLeuAlaAsnValLysAlaHisLeuThrGlnLeu

151015

SerThrIleAlaAlaAsnAsnGlyGlyAsnArgAlaHisGlyArgPro

202530

GlyTyrLysAlaSerValAspTyrValLysAlaLysLeuAspAlaAla

354045

GlyTyrThrThrThrLeuGlnGlnPheThrSerGlyGlyAlaThrGly

505560

TyrAsnLeuIleAlaAsnTrpProGlyGlyAspProAsnLysValLeu

65707580

MetAlaGlyAlaHisLeuAspSerValSerSerGlyAlaGlyIleAsn

859095

AspAsnGlySerGlySerAlaAlaValLeuGluThrAlaLeuAlaVal

100105110

SerArgAlaGlyTyrGlnProAspLysHisLeuArgPheAlaTrpTrp

115120125

GlyAlaGluGluLeuGlyLeuIleGlySerLysPheTyrValAsnAsn

130135140

LeuProSerAlaAspArgSerLysLeuAlaGlyTyrLeuAsnPheAsp

145150155160

MetIleGlySerProAsnProGlyTyrPheValTyrAspAspAspPro

165170175

ValIleGluLysThrPheLysAsnTyrPheAlaGlyLeuAsnValPro

180185190

ThrGluIleGluThrGluGlyAspGlyArgSerAspHisAlaProPhe

195200205

LysAsnValGlyValProValGlyGlyLeuPheThrGlyAlaGlyTyr

210215220

ThrLysSerAlaAlaGlnAlaGlnLysTrpGlyGlyThrAlaGlyGln

225230235240

AlaPheAspArgCysTyrHisSerSerCysAspSerLeuSerAsnIle

245250255

AsnAspThrAlaLeuAspArgAsnSerAspAlaAlaAlaHisAlaIle

260265270

TrpThrLeuSerSerGlyThrGlyGluProProThr

275280

<210>81

<211>299

<212>PRT

<213>Vibrioproteolyticus

<400>81

MetProProIleThrGlnGlnAlaThrValThrAlaTrpLeuProGln

151015

ValAspAlaSerGlnIleThrGlyThrIleSerSerLeuGluSerPhe

202530

ThrAsnArgPheTyrThrThrThrSerGlyAlaGlnAlaSerAspTrp

354045

IleAlaSerGluTrpGlnAlaLeuSerAlaSerLeuProAsnAlaSer

505560

ValLysGlnValSerHisSerGlyTyrAsnGlnLysSerValValMet

65707580

ThrIleThrGlySerGluAlaProAspGluTrpIleValIleGlyGly

859095

HisLeuAspSerThrIleGlySerHisThrAsnGluGlnSerValAla

100105110

ProGlyAlaAspAspAspAlaSerGlyIleAlaAlaValThrGluVal

115120125

IleArgValLeuSerGluAsnAsnPheGlnProLysArgSerIleAla

130135140

PheMetAlaTyrAlaAlaGluGluValGlyLeuArgGlySerGlnAsp

145150155160

LeuAlaAsnGlnTyrLysSerGluGlyLysAsnValValSerAlaLeu

165170175

GlnLeuAspMetThrAsnTyrLysGlySerAlaGlnAspValValPhe

180185190

IleThrAspTyrThrAspSerAsnPheThrGlnTyrLeuThrGlnLeu

195200205

MetAspGluTyrLeuProSerLeuThrTyrGlyPheAspThrCysGly

210215220

TyrAlaCysSerAspHisAlaSerTrpHisAsnAlaGlyTyrProAla

225230235240

AlaMetProPheGluSerLysPheAsnAspTyrAsnProArgIleHis

245250255

ThrThrGlnAspThrLeuAlaAsnSerAspProThrGlySerHisAla

260265270

LysLysPheThrGlnLeuGlyLeuAlaTyrAlaIleGluMetGlySer

275280285

AlaThrGlyAspThrProThrProGlyAsnGln

290295

<210>82

<211>30

<212>DNA

<213>Artificialsequence

<220>

<223>Cloningprimer

<400>82

atatataagcttatggcaggcggaagacac30

<210>83

<211>31

<212>DNA

<213>Artificialsequence

<220>

<223>Cloningprimer

<400>83

atatgcggccgcttacaaatgaagatattcc31

<210>84

<211>35

<212>DNA

<213>Artificialsequence

<220>

<223>PCRprimer

<400>84

atatatgcggccgcctagagccccaggtattcagc35

<210>85

<211>31

<212>DNA

<213>Artificialsequence

<220>

<223>PCRprimer

<400>85

atatctcgagtccatcgccaccatggtgagc31

<210>86

<211>31

<212>DNA

<213>Artificialsequence

<220>

<223>PCRprimer

<400>86

atatctcgagttacttgtacagctcgtccat31

<210>87

<211>41

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>87

atatgcggccgcatgtcgacgctccaaatggtgtagaacgc41

<210>88

<211>57

<212>DNA

<213>Artificialsequence

<220>

<223>PCRprimer

<400>88

atatgcggccgcttacttgtcatcgtcatccttgtaatccaaatgaagatattccaa57

<210>89

<211>57

<212>DNA

<213>Artificialsequence

<220>

<223>PCRprimer

<400>89

atatgcggccgcctacttgtcatcgtcatccttgtaatcgagccccaggtattcagc57

<210>90

<211>20

<212>DNA

<213>Artificialsequence

<220>

<223>PCRprimer

<400>90

gcctccagcatgaaagtctc20

<210>91

<211>20

<212>DNA

<213>Artificialsequence

<220>

<223>CAGATCTCCTTGGCCACAAT

<400>91

cagatctccttggccacaat20

<210>92

<211>20

<212>DNA

<213>Artificialsequence

<220>

<223>PCRprimer

<400>92

atgaaagcctctgcagcact20

<210>93

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>93

tggctactggtggtccttct20

<210>94

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>94

tcacctgctgctttaacgtg20

<210>95

<211>20

<212>DNA

<213>Artificialsequence

<220>

<223>PCRprimer

<400>95

atccctgacccatctctcct20

<210>96

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>96

atctccttgcagaggctgaa20

<210>97

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>97

agaagaggaggccagaggag20

<210>98

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>98

cacagaaatggccttgtgaa20

<210>99

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>99

ccaagcaggtcataggtggt20

<210>100

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>100

tcctttcatcctggaacctg20

<210>101

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>101

cgcctcttctgtttcacctc20

<210>102

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>102

aagcgctgtttgccagttat20

<210>103

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>103

cacacgtgaggcgctattta20

<210>104

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>104

gtcaacagatcctccccaga20

<210>105

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>105

cagcatttctgcctttgtga20

<210>106

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>106

aggtggagagcctgaggaat20

<210>107

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>107

ctcgggtcctacttgtcagc20

<210>108

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>108

aagcgaggttctcgttctga20

<210>109

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>109

tgacctcttgctctccctgt20

<210>110

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>110

cttcaagctctcctgctgct20

<210>111

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>111

cgaccctgacttcctggtta20

<210>112

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>112

gctcatcggctgttggtatt20

<210>113

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>113

ataagcaggtggagcattgg20

<210>114

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>114

atgcttcggaaactggacat20

<210>115

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>115

atggttcgatgcagctttct20

<210>116

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>116

tacggcgtaatcctggaaac20

<210>117

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>117

attgtgcatgctgctttgag20

<210>118

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>118

ccgaaacacagtggaaggtt20

<210>119

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>119

tctgtgaaggtgtgcaggag20

<210>120

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>120

ggttcctttcttccctccag20

<210>121

<211>20

<212>DNA

<213>artificialsequence

<220>

<223>PCRprimer

<400>121

aaccaaagccaccagtgttc20

Claims

1. screening can suppress the method for the compound of the enzymic activity of at least one polypeptide, described polypeptide can be optionally glycosylated, and this polypeptide (a) is made up of the aminoacid sequence of the maturation protein shown in any one of SEQIDNO:11-18, described method is included in the suitable substrates of maturation protein and this maturation protein described in incubation when there is one or more test compounds or its salt, measure the enzymic activity of this maturation protein, this active is compared with determined commeasurable activity when there is not test compounds, and select one or more test compounds reducing described enzymic activity,

Wherein said test compounds is competitiveness enzyme inhibitor.

2. screening does not suppress the method for the selectivity QC inhibitor of the enzymic activity of at least one polypeptide, described polypeptide can be optionally glycosylated, and this polypeptide (a) is made up of the aminoacid sequence of the maturation protein shown in any one of SEQIDNO:11-18, maturation protein and suitable substrates described in incubation when described method is included in one or more inhibitor or its salt of there is QC, measure the enzymic activity of this maturation protein, this is active in determined commeasurable expression activitiy when there is not described QC inhibitor, and select the compound of the enzymic activity not reducing described maturation protein,

Wherein said selectivity QC inhibitor is competitiveness enzyme inhibitor.

3. the method for claim 1 or 2, wherein said maturation protein is made up of the aminoacid sequence of the maturation protein of one of SEQIDNO:11-18.

4. screening does not suppress the method for the selectivity QPCTL-inhibitor of the enzymic activity of QC, QC described in incubation when described method is included in one or more inhibitor or its salt of there is QPCTL, measure the enzymic activity of QC, this is active in determined commeasurable expression activitiy when there is not described QPCTL inhibitor, and select the compound of the enzymic activity not reducing described QPCTL albumen

Wherein said selectivity QPCTL inhibitor is competitiveness enzyme inhibitor.