CN101223283A - Compositions and methods for treating malaria with cupredoxin and cytochrome - Google Patents

Abstract

The present invention relates to cupredoxin and cytochrome and their use, separately or together, to inhibit the spread of parasitemia in mammalian red blood cells and other tissues infected by the malaria parasite, and in particular the parasitemia of human red blood cells by P. falciparum. The invention provides isolated peptides that are variants, derivatives or structural equivalents of cupredoxins or cytochrome c, and compositions comprising cupredoxins and/or cytochrome c, or variants, derivatives or structural equivalents thereof, that are useful for treating or preventing malaria infection in mammals. Further, the invention provides methods to treat mammalian patients to prevent or inhibit the growth of malarial infection in mammals. The invention also provides methods to prevent the growth of malaria infection in insect vectors.

Description

Composition and method with cupredoxin and cytopigment treatment malaria

Related application

The application's requirement _ _ _ _ day common U.S. Provisional Patent Application of submitting to that is entitled as " Compositions andMethods for Treating HIV Infection with Cupredoxin and Cytochrome c " _ _ _ _, the U.S. Provisional Patent Application 60/780 that on March 10th, 2006 submitted to, 868, the U.S. Provisional Patent Application 60/682 that on May 20th, 2005 submitted to, 813, the U.S. Patent application 11/244 that on October 6th, 2005 submitted to, the U.S. Provisional Patent Application 60/680 that on May 13rd, 105 and 2005 submitted to, 500 right of priority, this U.S. Patent application 11/244,105 require the U.S. Provisional Patent Application 60/616 of submission on October 7th, 2004,782 right of priority, and the application is the U.S. Patent application of submitting on November 11st, 2,003 10/720,603 parts continue, this U.S. Patent application 10/720,603 require the U.S. Provisional Patent Application 60/414 of submission on August 15th, 2003,550 preferential be for the time being the U.S. Patent application of submitting on November 15th, 2,002 10/047,710 part continues, this U.S. Patent application 10/047,710 require the right of priority of the U.S. Provisional Patent Application 60/269,133 of submission on February 15 calendar year 2001.These complete contents in first to file all are incorporated herein by reference.

The statement of governmental interests

The application's theme obtains from National Institutes of Health (NIH), Bethesda, Maryland, the support of U.S.A. (AI 45541, CA09432 and N01-CM97567 for subsidy AI 16790-21, ES 04050-16).Government may have some right in the present invention.

Technical field

The present invention relates to cupredoxin and cytopigment and they alone or in combination in the parasitemia that suppresses plasmodium (malaria parasite), particularly suppress the application in the parasitemia of the plasmodium falciparum (Plasmodium falciparum) in the mammalian erythropoietin.The invention still further relates to the variant and the derivative of cupredoxin and cytopigment, they have kept the plasmodial parasitemia ability that suppresses.At last, the invention provides the method that suppresses the propagation of malaria infection in the insect vector.

Background technology

World crowd's about 1/4th is exposed in the risk of malaria, surpasses a million people every year and dies from malaria.In four kinds of plasmodiums of infected person, two main kinds are plasmodium falciparum and Plasmodium vivax (P.vivax).

Plasmodium falciparum blood stage schizont utilizes the kinds of surface protein binding and parasitizes (Cowman et al., FEBS Lett.476:84-88 (2000) in the red corpuscle; Baum et al., J.Biol.Chem.281:5197-5208 (2006)), its major antigen member is called as schizont surface protein 1 (MSP1), is the albumen of a kind of 195kDa.MSP1 is present in plasmodial all red corpuscle invasive kinds, and it anchors to the schizont surface by glycosyl-phosphatidylinositols key.Early stage at the red corpuscle invasion procedure, after discharging from infected red corpuscle soon, schizont MSP1 albumen experience proteolytic cleavage, produce the terminal split product MSP1-42 of C-, it experiences cracking for the second time subsequently, produce 11kDa peptide MSP1-19, it remains adhered to the parasite surface when parasite enters red corpuscle.The formation of split product MSP1-19 is very important for parasitic successfully the intrusion, because inhibition that its proteolysis is formed or the neutralization by monoclonal antibody have stoped parasite to enter red corpuscle (Blackman et al., J.Exptl., Med.180:389-393 (1994)).

The MSP1-19 peptide is a kind of most important obtainable malaria vaccine material standed for.From the MSP1-19 specific antibody and the antigen-reactive of malaria resistance human serum, and it comprises main red corpuscle intrusion inhibition part (Holder ﹠amp; Riley, Parasitol.Today, 12:173-174 (1996); O ' Donnell et al., J.Expt.Med.193:1403-1412 (2001)).From the serum of the donor in malaria endemy zone usually performance to the powerful antibody reactivity of Pf MSP1-19.(Nwuba et al.，Infect.Immun.70：5328-5331(2002))。

Monoclonal antibody (mAb) G17.12 produces at reorganization PfMSP1-19, and discern it in the lip-deep epi-position of parasite, show that antigenic this district is come-at-able (Pizarro et al., J.Mol.Biol.328:1091-1103 (2003)) on natural MSP1 polypeptide complex.Enjoyably, erythrocyte in vitro is invaded experiment and is shown, in the presence of G17.12, even under the concentration of 200 μ g/ml, infects not being suppressed, and does not suppress the secondary processing of MSP1 at external G17.12.The same.Thereby also verified blocking-up invade the combination of inhibiting antibody promote the parasite survival antibody have (Guevara Patino an etal., J.Expt.Med.186:1689-1699 (1997)), the existence of this antibody may invade be responsible for the red corpuscle that G17.12mAb can not suppress M.falciparum.

Cerebral malaria is a kind of owing to being limited to the rare of brain plasmodium falciparum intrusion capillaceous but lethal infection by parasitic erythrocytic detaining, and it normally can not be treated, and reaches the brain kapillary because most drug can not be passed through hemato encephalic barrier.The red corpuscle of falciparum infection is to the interaction mediation of brain adhesion capillaceous by the ICAM-1 and the CD36 of capillary endothelial cells surface expression in the parasite part Pf Emp-1 protein family of expressing on the infected erythrocyte surface and the cerebrovascular.(Smith et al.，Proc.Natl.Acad.Sci.USA 97：1766-1771(2000)；Franke-Fayard et al.，Proc.Natl.Acad.Sci.USA 102，11468-11473(2005))。

Though some medicines for example chloroquine of target protoheme detoxification pathways are used to treat malaria, parasite increases the resistance of medicine and the mosquito media incidence to resistance to insecticides.The protoheme polymerization that chloroquine antagonism parasite inductive HRP (rich histidine protein) is mediated is because the protoheme monomer is highly toxic to plasmodium.The polymerization of protoheme can be detoxified, and this is reversed by chloroquine.Another kind of medicine Artemisinin is effective to chloroquine resistance plasmodium falciparum in the cerebral malaria.Artemisinin combines protoheme with sphaeroprotein in the oxyphorase and forms adducts, its in conjunction with HRP in case the polymerization of hemostasis red pigment.In Africa and the fearful disease of the special popular in Asia, be badly in need of finding new medicine for this.The current trial of drug development is devoted to decode the proteic molecule modeling of complete parasite genome sequence, plasmodium and to the searching of new drug target.

Summary of the invention

The present invention relates to the application that cupredoxin and cytopigment and they suppress the propagation of the HRBC parasitemia of parasitemia, particularly plasmodium falciparum in its hetero-organization that mammalian erythropoietin and plasmodium infect separately or together.

One aspect of the present invention is isolating peptide, and it is variant, derivative or the structural equivalents of cupredoxin or cytopigment; And it can suppress plasmodial time multiplexed cell system in the HRBC of malaria infection.

Another aspect of the present invention is isolating peptide, and it is variant, derivative or the structural equivalents of cupredoxin; And it can be in conjunction with the albumen that is selected from PfMSP1-19 and PfMSP1-42.

Another aspect of the present invention is isolating peptide, and it is variant, derivative or the structural equivalents of cupredoxin or cytopigment, and it can suppress the malarial parasite mass formed by blood stasis in the red corpuscle of malaria infection.Especially, described isolating peptide can suppress the malarial parasite mass formed by blood stasis in the HRBC of falciparum infection.In some embodiments, described cupredoxin is azurin, false azurin, plastocyanin, iron sulphur bacterium azurin (rusticyanin), Laz or the green element (auracyanin) that deflects of orange.Especially, described cupredoxin can be iron sulphur bacterium azurin, azurin or Laz.In some embodiments, described cupredoxin is from Pseudomonas aeruginosa (Pseudomonas aeruginosa), Alcaligenes faecalis (Alcaligenes faecalis), Achromobacter xylosoxidans (Achromobacter xylosoxidan), bordetella bronchiseptica (Bordetella bronchiseptica), methylomonas (Methylomonas sp.), Neisseria meningitidis (Neisseria meningitidis), Diplococcus gonorrhoeae (Neisseria gonorrhea), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas chlororaphis (Pseudomonaschlororaphis), xyllela fastidiosa (Xylella fastidiosa) or Vibrio parahaemolyticus (Vibrioparahaemolyticus).Especially, described cupredoxin can be from thiobacillus ferrooxidant (Thiobacillus ferrooxidans), Pseudomonas aeruginosa, Diplococcus gonorrhoeae or Neisseria meningitidis.

In these other embodiments on the one hand, described cytopigment are cytochrome c or cytopigment f.Especially, described cytochrome c can be from people or Pseudomonas aeruginosa.Described cytopigment f can be from cyanobacteria (cyanobacteria).

In these other embodiments on the one hand, described isolating peptide is the clipped form that is selected from the peptide of SEQ ID NO:1-20 and 22.In some embodiments, SEQ ID NO:1-20 or 22 and the sequence of described isolating peptide have at least 90% aminoacid sequence homogeny.

In these some embodiments on the one hand, described isolating peptide is the clipped form of cupredoxin or cytopigment.In some embodiments, described isolating peptide surpasses about 10 residues and is no more than about 100 residues.Described isolating peptide can comprise azurin residue 36-89.Perhaps, described isolating peptide can be made up of azurin residue 36-89.Perhaps, described isolating peptide can comprise cupredoxin and residue azurin 36-89 equivalence.

In these other embodiments on the one hand, merge in the H.8 district of described isolating peptide and Laz.In another embodiment, the described isolating peptide structural equivalents that is monoclonal antibody G17.12.

Another aspect of the present invention is a kind of composition, it is contained at least a cupredoxin, cytopigment or isolating peptide in the pharmaceutical composition, and described isolating peptide is to suppress the cupredoxin of the malarial parasite mass formed by blood stasis in the red corpuscle of malaria infection or variant, derivative or the structural equivalents of cytopigment.Especially, can prepare described pharmaceutical composition for intravenous administration.Described composition can comprise another kind of antimalarial drug or anti-HIV medicine.

In some embodiments of described composition, described cupredoxin is from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidans, bordetella bronchiseptica, methylomonas, Neisseria meningitidis, Diplococcus gonorrhoeae, Pseudomonas fluorescens, Pseudomonas chlororaphis, xyllela fastidiosa or Vibrio parahaemolyticus.Especially, described cupredoxin can be from thiobacillus ferrooxidant, Pseudomonas aeruginosa, Diplococcus gonorrhoeae or Neisseria meningitidis.

In some embodiments of described composition, described cytopigment are cytochrome c or cytopigment f.Especially, described cytochrome c can be from people or Pseudomonas aeruginosa.Described cytopigment f can be from cyanobacteria.In some embodiments, described cupredoxin or cytochrome c are SEQ ID NO:1-20 or 22.

Another aspect of the present invention is the method that treatment suffers the patient of plasmodium infection, and it is undertaken by the composition of the present invention to described patient's effective dosage.In specific embodiment, described peptide suppresses the malarial parasite mass formed by blood stasis in described patient's the HRBC of malaria infection.In some embodiments, described plasmodium is Plasmodium vivax or plasmodium falciparum.In some embodiments, described patient is infected by HIV in addition.In some embodiments, described composition is with the second composition administration, and described second composition can contain antimalarial drug and/or anti-HIV medicine.In some embodiments, composition of the present invention administration in 0 minute to 12 hours of administration second composition.In some embodiments, with composition oral administration of the present invention, suction, intravenously, intramuscularly or through subcutaneous administration in described patient; Especially, can be with described composition through intravenous administration in described patient.

Another aspect of the present invention is the doubtful patient's who contacts with plasmodium of treatment a method, and it comprises the composition of the present invention to described patient's effective dosage.

Another aspect of the present invention is the method for the malaria of prevention in the Mammals, and it comprises the composition of the present invention of insect vector administration one tittle in carrying plasmodial insect vector colony.In some embodiments of this method, described composition oral administration is delivered medicine to described insect vector.

These and other aspects of the present invention, benefit and feature will become clear from the following drawings and embodiment.

The accompanying drawing summary

Fig. 1. Fig. 1 display surface plasma resonance is in conjunction with titration, and it provides azurin, the H.8-interaction of azurin (H.8-Az), Laz and GST-azurin (GST-Azu) construct and MSP 1-19 and 1-42.(A) binding curve shows the interaction of the MSP1-19 (MSP1-19-CM5) on azurin and its analogue and the golden susceptor chip that is immobilized in carboxyl methyl dextran bag quilt.Combine with the concentration dependent of MSP1-19 by measuring azurin, and combination degree is estimated as the function of the balance resonant reaction value of measuring with resonance units (RU) at susceptor surface injection different concns (0.05-300nM).Though H.8-Az with Laz than some combination more consumingly of azurin, with GST or H.8-GST do not observe combination.(B) externally combine titration with what carry out immobilization MSP1-42 and azurin and its analogue with the similar fashion that is used for MSP1-19 shown in (A).By with data fitting to Req=Rmax/ (1+ (Kd/C)), measure relevant binding affinity with the fitting of a curve of the data point in the interface chart.MSP1-19 in conjunction with the Kd value is: 32.2 ± 2.4nM (azurin), 26.2 ± 2.4nM (Laz), 11.8 ± 0.3nM (H.8-Az) to the MSP1-42 bonded is: 54.3 ± 7.6nM (azurin), 45.6 ± 2.4nM (Laz) and 14.3 ± 1.7nM (H.8-Az).(C) in the interactional identification that shows GST-Azu 36-128 and GST-Azu 36-89 and MSP1-19 in conjunction with titration of the GST-Azu of MSP1-19-CM5 sensor surface fusion rotein.Do not observe combination with GST or GST-Azu 88-113.

Fig. 2. Fig. 2 shows the azurin of as directed different concns, H.8-azurin (H.8-Az) and Laz be to the inhibition of Plasmodium falciparum parasites mass formed by blood stasis (the entozoal growth of RBC).In these experiments, in the albumen existence of different concns or not, infect normal red corpuscle with schizont, be incubated overnight, come the entozoal number count of pair cell by thin blood smear and Giemsa staining.

Fig. 3. Fig. 3 shows the bonded surface plasma resonance binding curve of ICAM (ICAM-1, ICAM-2, ICAM-3 and NCAM, illustration) and immobilization azurin.Owing to, CM5 is added (1mg/ml CM5 adds in the HBS-EP damping fluid) in the electrophoretic buffer as elutriant to a large amount of non-specific binding of naked Au-CM5 chip.The selectivity identification of azurin and ICAM-3 is significant, and bonding strength is 19.5 ± 5.4nM, and azurin is not discerned with ICAM-1 or ICAM-2 selectivity.Shown in illustration, NCAM and azurin bonded Kd are 20 ± 5.0nM.

The sequence brief description

SEQ ID NO:1 is the aminoacid sequence from the azurin of Pseudomonas aeruginosa.

SEQ ID NO:2 is the cytochrome c from Pseudomonas aeruginosa ₅₅₁Aminoacid sequence.

SEQ ID NO:3 is the aminoacid sequence from the Laz of Neisseria meningitidis MC58.

SEQ ID NO:4 is the aminoacid sequence from the plastocyanin of bedding seat algae (Phormidium laminosum).

SEQ ID NO:5 is the aminoacid sequence from the iron sulphur bacterium azurin of thiobacillus ferrooxidant (having a liking for the ferrous thiobacillus Acidithiobacillus of acid oxidase ferrooxidans).

SEQ ID NO:6 is the aminoacid sequence from the false azurin of driffractive ring achromobacter (Achromobacter cycloclastes).

SEQ ID NO:7 is the aminoacid sequence from the azurin of Alcaligenes faecalis.

SEQ ID NO:8 is the aminoacid sequence from the azurin of Achromobacter xylosoxidans subspecies denitrificans I.

SEQ ID NO:9 is the aminoacid sequence from the azurin of bordetella bronchiseptica.

SEQ ID NO:10 is the aminoacid sequence from the azurin of methylomonas J..

SEQ ID NO:11 is the aminoacid sequence from the azurin of Neisseria meningitidis Z2491.

SEQ ID NO:12 is the aminoacid sequence from the azurin of Pseudomonas fluorescens.

SEQ ID NO:13 is the aminoacid sequence from the azurin of Pseudomonas chlororaphis.

SEQ ID NO:14 is the aminoacid sequence from the azurin of xyllela fastidiosa 9a5c.

SEQ ID NO:15 is the aminoacid sequence from the star cyanin (stellacyanin) of cucumber (Cucumis sativus).

SEQ ID NO:16 is from the orange green orange green aminoacid sequence that deflects plain A of subduing bacterium (Chloroflexus aurantiacus).

SEQ ID NO:17 is from the orange green orange green aminoacid sequence that deflects plain B of subduing bacterium.

SEQ ID NO:18 is the proteic aminoacid sequence of cucumber alkalescence from cucumber.

SEQ ID NO:19 is from human Homo sapiens) the aminoacid sequence of cytochrome c.

SEQ ID NO:20 is the aminoacid sequence from the cytopigment f of cyanobacteria bedding seat algae.

SEQ ID NO:21 is the H.8 aminoacid sequence in district from Diplococcus gonorrhoeae F62.

SEQ ID NO:22 is the aminoacid sequence from the Laz of Diplococcus gonorrhoeae F62.

SEQ ID NO:23 is the forward primer that is used for the Laz encoding gene (laz) of pcr amplification Diplococcus gonorrhoeae.

SEQ ID NO:24 is the reverse primer that is used for the Laz encoding gene (laz) of pcr amplification Diplococcus gonorrhoeae.

SEQ ID NO:25 is the segmental forward primer of 3.1kb that is used for pcr amplification pUC18-laz.

SEQ ID NO:26 is the segmental reverse primer of 3.1kb that is used for pcr amplification pUC18-laz.

SEQ ID NO:27 is the segmental forward primer of 0.4kb that is used for pcr amplification pUC19-paz.

SEQ ID NO:28 is the segmental reverse primer of 0.4kb that is used for pcr amplification pUC19-paz.

SEQ ID NO:29 is the forward primer of pGST-azu 36-128.

SEQ ID NO:30 is the reverse primer of pGST-azu 36-128.

SEQ ID NO:31 is the forward primer of pGST-azu 36-89.

SEQ ID NO:32 is the reverse primer of pGST-azu 36-89.

SEQ ID NO:33 is the forward primer of pGST-azu 88-113.

SEQ ID NO:34 is the reverse primer of pGST-azu 88-113.

SEQ ID NO:35 is used for site-directed mutagenesis to be used to prepare the oligonucleotide of pGST-azu 88-113.

SEQ ID NO:36 is used for site-directed mutagenesis to be used to prepare the oligonucleotide of pGST-azu 88-113.

Embodiment

Definition

As used herein, term " cell " comprises the odd number and the plural number of this term, unless be described as " individual cells " especially.

As used herein, term " polypeptide ", " peptide " and " albumen " make the polymkeric substance that is used for referring to amino-acid residue interchangeably.This term is applicable to such aminoacid polymers, and wherein one or more amino-acid residues are corresponding naturally occurring amino acid whose artificial chemical analogs.This term also is applicable to naturally occurring aminoacid polymers.Term " polypeptide ", " peptide " and " albumen " also comprise modified polypeptides ", " peptide " and " albumen ", it includes but not limited to γ carboxylation, hydroxylation and the ADP ribosylation of glycosylation, lipid connection, sulfation, glutaminic acid residue.It being understood that polypeptide is always fully linear.For example, can be ramose as the polypeptide as a result of ubiquitinization, they can be annular (being with or without branch), generally are the results of translation back incident, comprise the incident that natural processing incident and the manual operation that is existed by non-natural cause.Annular, ramose and branch's annular polypeptide can be by untranslated natural process and synthesize by synthetic method completely.

As used herein, term " pathologic conditions " comprises with respect to normal dissection and physiological deviation, it constitutes the infringement of living animal or its a part of standard state, its blocking-up or changed the performance of body function, and be to various factors (for example malnutritive, industrial hazard or weather), to specific infection material (as worm, parasitic protozoa, bacterium or virus), to the inherent defect (unusual as genetics) of organism or to the reaction of the combination of these factors.

As used herein, use " illness " to comprise with respect to normal dissection and physiological departing from, it constitutes the infringement of living animal or its a part of standard state, the performance of its blocking-up or change body function.

As used herein, term " suffers from ", " suffering " comprise current performance pathologic conditions symptom, have pathologic conditions, be in the recovery of pathologic conditions or from pathologic conditions, recovered even without visible symptom.

As used herein, term " parasitemia " comprises that parasite wherein is present in the illness in blood and its hetero-organization, is meant the illness that the parasite that is with or without clinical symptom exists especially.

As used herein, term " inhibition of parasitemia " is meant the reduction or the attenuating of the speed of growth that parasite exists in mammalian.Inhibition is any reduction or the attenuating of comparing the significant speed of growth of statistics with control treatment.

As used herein, term " treatment " comprises progress or the severity that prevents, reduces, stops or reversing illness or the symptom relevant with the illness that will treat.Thereby term " treatment " comprises suitable medicine, treats and/or prevents administration.

As used herein, " anti-malarial activity " comprises any activity that reduces infectivity, breeding or suppress the development of plasmodium life cycle." anti-malarial activity " comprises by suppress the growth of malaria infection with observed all methods of present antimalarial drug.

As used herein, term " antimalarial drug " is meant the medicine that has the anti-malarial activity, can be used for reducing infectivity, breeding or suppress the development of plasmodium life cycle.

As used herein, term " anti-HIV medicine " be meant medicine with HIV (human immunodeficiency virus)-resistant activity, infect by the HIV of described medicine in any way Mammals and to be lowered or to stop increase in its human body, described any way to include but not limited to suppress that HIV is genomic duplicates, suppresses the synthetic and/or assembling of HIV coat protein and suppress HIV to enter the cell that does not infect.

As used herein, term " pure basically " when being used to modify term polypeptide or other compounds, is meant a peptide species or compound, for example separates the polypeptide from the substratum of growing, and its form is not for basically or the active inhibition reagent that undopes.Term " pure basically " be meant with the dry weight metering be separate part at least about 75% compound, or " 75% is pure basically ".More specifically, term " pure basically " is meant and is at least about 85% compound with the dry weight basis active compound, or " 85% is pure basically ".The most particularly, term " pure basically " is meant and is at least about 95% compound with the dry weight basis active compound, or " 95% is pure basically ".Basically pure cupredoxin or cytochrome c ₅₅₁Or its variant or derivative can be used in combination with one or more other pure basically compound or other isolating cupredoxins or cytopigment.

Phrase " isolating ", " purifying " or " biology is pure " are meant a kind of material, and it contains the component of following this material usually as existing basically or hardly in native state.Thereby, preferably do not contain the common material relevant in the in situ environment of described peptide according to isolating peptide of the present invention with described peptide." isolating " district is meant the zone of the whole sequence that does not comprise polypeptide, and described zone is from described polypeptide." isolating " nucleic acid, albumen or its respective segments shift out from its internal milieu basically, make it to operate by those skilled in the art, in expression vector, and obtain albumen or protein fragments such as but not limited to nucleotide sequencing, restrictive diges-tion, site-directed mutagenesis and the subclone of nucleic acid fragment with pure basically amount.

Be meant the aminoacid sequence variant as this paper for the used term of peptide " variant ", it is compared with wild type peptide can have amino acid alternative, that lack or insert.Variant can be the clipped form of wild type peptide.Thereby variant peptides can produce by the genetic manipulation to coding said polypeptide.By essentially consist or the feature that changes described polypeptide, still do not change at least some of its primary activity, can produce variant.For example, " variant " of azurin can be the azurin of sudden change, and it has kept the ability that it suppresses parasitemia in the HRBC of malaria infection.In some cases, use alpha-non-natural amino acid for example ε-(3,5-phenodiazine benzoyl)-Lys residue synthesize variant peptides.(Ghadiri & Fernholz，J.Am.Chem.Soc，112：9633-9635(1990))。In some embodiments, compare with wild type peptide, described variant have be no more than 20 substitute, disappearance or the amino acid that insert.In some embodiments, compare with wild type peptide, described variant have be no more than 15,14,13,12 or 11 substitute, disappearance or the amino acid that insert.In some embodiments, compare with wild type peptide, described variant have be no more than 10,9,8 or 7 substitute, disappearance or the amino acid that insert.In some embodiments, compare with wild type peptide, described variant have be no more than 6 substitute, disappearance or the amino acid that insert.In some embodiments, compare with wild type peptide, described variant have be no more than 5 or 4 substitute, disappearance or the amino acid that insert.In some embodiments, compare with wild type peptide, described variant have be no more than 3,2 or 1 substitute, disappearance or the amino acid that insert.

As used herein, term " amino acid " is meant and comprises any naturally occurring or non-natural amino acid moiety that exist or the synthetic amino-acid residue, promptly comprise through one, two, three or more carbon atom any part of direct-connected at least one carboxyl of a general carbon atom and at least one amino residue.

Be meant peptide as this paper for the used term of peptide " derivative " derived from target peptide.The chemically modified form of deriving and comprising peptide makes described peptide still keep some its primary activities.For example, " derivative " of azurin can be the azurin of chemically modifying, and it has kept the ability that it suppresses parasitemia in the HRBC of malaria infection.Interested chemically modified includes but not limited to amidation, acetylize, sulfation, polyoxyethylene glycol (PEG) modification, phosphorylation or the glycosylation of peptide.In addition, the deutero-peptide can be the fusion of polypeptide or its fragment and compound, and described compound is such as but not limited to another kind of peptide, drug molecule or other treatment agent or medicine or detectable probe.

Term " aminoacid sequence homogeny per-cent (%) " is defined as when with two sequence parallelisms, the per-cent of the amino-acid residue identical with amino-acid residue in the candidate sequence in the polypeptide.In order to determine amino acid homogeny %,, if necessary, introduce breach to obtain maximum sequence homogeny % with the sequence parallelism; Conservative replacement is not considered to the part of sequence homogeny.The aminoacid sequence parallelism method of determining homogeny per-cent is well known to a person skilled in the art.Use for example BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software parallelism peptide sequence of the obtainable computer software of the common public.In specific embodiment, use Blastp (can be from state-run biotechnology information center, Bethesda MD obtains), it uses long complicacy filter (long complexity filter) default parameters expection 10, word length 3, have 11 and extend 1.

When aminoacid sequence during by parallelism, given aminoacid sequence A with and or with respect to the aminoacid sequence homogeny % of given aminoacid sequence B (perhaps it can be called given aminoacid sequence A, its with and or have or comprise certain aminoacid sequence homogeny % with respect to given aminoacid sequence B) may be calculated:

Aminoacid sequence homogeny %=X/Y*100

Wherein

X is a total number of atnino acid of making identical match by the parallelism score of the sequence parallelism program of A and B or algorithm, and

Y is the sum of amino-acid residue among the B.

If aminoacid sequence A is uneven in length in the length of aminoacid sequence B, then the aminoacid sequence homogeny % of A and B will be not equal to the aminoacid sequence homogeny % of B and A.In the time will comparing than long sequence and than short sequence, the sequence of weak point will be " B " sequence, except as otherwise noted.For example, when with the peptide of brachymemma and corresponding wild type polypeptide relatively the time, the peptide of brachymemma will be " B " sequence.

" treatment significant quantity " be effectively prevent or particular disorder, pathology or other situations of the individuality that will treat of slowing down in existing symptom development or alleviate the amount of existing symptom partially or completely.The treatment significant quantity fixes in those skilled in the art's the limit of power really.

Summary of the invention

The invention provides composition and method, it uses cupredoxin and/or cytopigment with the mammalian erythropoietin that suppresses malaria infection and the body tissue parasitemia of cerebral tissue and osseous tissue for example.

Known in the past, belong to several bacterial oxidations reduction albumen that contain blue copper protein family or be called iron content (protoheme) protein family of cytopigment that are called cupredoxin and all enter in the mammalian cell that comprises cancer cells, and apoptosis-induced cell death or the G1 by the cell cycle stagnate and cause growth-inhibiting.(Yamada et al.，Cell Cycle3：752-755(2004)；Yamada et al.，Cell Cycle 3：1182-1187(2004))。Single bacterioprotein is for example by exquisite cupredoxin azurin or the cytochrome c that produces of Pseudomonas aeruginosa ₅₅₁, can show hydrophobic activity based on them.Therefore, wild-type (wt) azurin (Yamada et al. apoptosis-induced in mouse J774 cell, and mutant M44KM64E azurin causes that in the G1 phase cell cycle suppresses in the J774 cell Infection andImmunity 70:7054-7062 (2002)).(Yamada et al.，PNAS101：4770-4775(2004))。On the contrary, wt cytochrome c ₅₅₁In the J774 cell, cause the cell cycle inhibition in the G1 phase, and mutant V23DI59E cytochrome c ₅₅₁Apoptosis-induced.(Hiraoka et al.，PNAS 101：6427-6432(2004))。

According to the present invention, astoundingly, now to know, cupredoxin and cytopigment are in the external parasitemia that will suppress plasmodium plasmodium falciparum in the HRBC.Especially, cupredoxin azurin and Laz suppress the parasitemia about 50% and about 75% of plasmodium falciparum respectively.Referring to embodiment 6.In addition, iron sulphur bacterium azurin and cytochrome c and f suppress parasitemia 20-30%.Referring to embodiment 1.In addition, now know, when with the Pf MSP1-19 fragment compound tense of the MSP1 surface protein of plasmodium falciparum, the Fab fragment that azurin has with the G17.12 mouse monoclonal antibody has recognizable structural homology.Referring to embodiment 2.Though the mode that suppresses is not limited to any mode, think that azurin can be by suppressing the parasitemia of plasmodium falciparum with the MSP1 protein-interacting on parasite surface.

Astoundingly, it is now know that, azurin and Laz all external in conjunction with PfMSP1-19 and PfMSP1-42 plasmodium falciparum surface proteins.In addition, it is now know that, and azurin amino-acid residue 36-89 combines required with PfMSP1-19 and PfMSP1-42.In addition, it is now know that, improved from the H.8 structural domain of the Laz of Diplococcus gonorrhoeae the azurin that merges and PfMSP1-19 combine and to the inhibition of the parasitemia of plasmodium falciparum.Referring to embodiment 5 and 6.

Because the attach structure homology between cupredoxin considers that other cupredoxin will have and azurin, iron sulphur bacterium azurin or the identical anti-malarial activity of Laz.In some embodiments, described cupredoxin is but is not limited to azurin, false azurin, plastocyanin, green element, Laz or the iron sulphur bacterium azurin of deflecting of orange.In specific embodiment, described cupredoxin is Laz, azurin or iron sulphur bacterium azurin.In other embodiments, described cupredoxin is from pathogenic bacteria.In more specific embodiment, described cupredoxin is an azurin.In specific especially embodiment, described azurin is from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidans subspecies denitrificans I, bordetella bronchiseptica, methylomonas, Neisseria meningitidis Z2491, Pseudomonas fluorescens, Pseudomonas chlororaphis, xyllela fastidiosa 9a5 or Vibrio parahaemolyticus.In the most specific embodiment, described azurin is from Pseudomonas aeruginosa.In other specific embodiments, described cupredoxin comprises the aminoacid sequence of SEQ ID NO:1,2-18 or 21.

According to the present invention, know the Pseudomonas aeruginosa cytochrome c ₅₅₁, human cytochrome c and bedding seat frustule pigment f will suppress the parasitemia in the HRBC of malaria infection.In specific embodiment, described cytopigment are the cytochrome cs from Pseudomonas aeruginosa ₅₅₁, human cytochrome c or cytopigment f.In other specific embodiments, described cytopigment comprise SEQ ID NO:2,19 or 20 aminoacid sequence.

Because the structural homology between cytochrome c considers that other cytopigment will have and the Pseudomonas aeruginosa cytochrome c ₅₅₁The anti-malarial activity identical with human cytochrome c.In some embodiments, described cytopigment are from pathogenic bacteria.In another specific embodiment, the parasitemia in the red corpuscle of described cytopigment inhibition malaria infection, more particularly, the parasitemia in the HRBC.In another specific embodiment, described cytopigment suppress mammalian cancer cells, more particularly the cell cycle of J774 cell progress.

Composition of the present invention

The invention provides peptide, it is variant, derivative or the structural equivalents of cupredoxin or cytopigment.In some embodiments, described peptide is pure basically.In other embodiments, described peptide is isolating.In some embodiments, described peptide is less than total length cupredoxin or cytopigment, and has kept some functional characteristics of cupredoxin or cytopigment.In some embodiments, described peptide has kept the ability of parasitemia in the red corpuscle that suppresses malaria infection, more particularly, suppresses the ability of falciparum infection in the HRBC.In specific embodiment, described cytopigment are Pseudomonas aeruginosa cytochrome cs ₅₅₁, human cytochrome c or cyanobacteria cytopigment f, SEQ ID NO:2,19 and 20 in particular.In another specific embodiment, described peptide does not more particularly cause immune response in the people not in Mammals.

The present invention also provides the composition that comprises at least a peptide, and described peptide is variant, derivative or the structural equivalents of cupredoxin, cytopigment or cupredoxin or cytopigment.The present invention also provides the composition that comprises at least a peptide, and described peptide is variant, derivative or the structural equivalents of cupredoxin or cupredoxin.The present invention also provides the composition that comprises at least a peptide, and described peptide is variant, derivative or the structural equivalents of cytopigment or cytopigment.In other embodiments, described composition is made up of described peptide basically.The present invention also provides the composition that is contained at least a peptide in the pharmaceutical composition, and described peptide is variant, derivative or the structural equivalents of cupredoxin, cytopigment or cupredoxin or cytopigment.

Because the attach structure homology between the cupredoxin considers that other cupredoxins will have the anti-malarial activity identical with the Pseudomonas aeruginosa azurin for the parasitemia in the red corpuscle that suppresses malaria infection.In some embodiments, described cupredoxin is but is not limited to azurin, false azurin, plastocyanin, iron sulphur bacterium azurin, Laz or the green element that deflects of orange.In specific especially embodiment, described cupredoxin is from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidans subspecies denitrificans I, bordetella bronchiseptica, methylomonas, Neisseria meningitidis Z2491, Diplococcus gonorrhoeae, Pseudomonas fluorescens, Pseudomonas chlororaphis, xyllela fastidiosa 9a5 or Vibrio parahaemolyticus.In very specific embodiment, described cupredoxin is the azurin from Pseudomonas aeruginosa.In other specific embodiments, described cupredoxin comprises the aminoacid sequence of SEQ ID NO:1,3-18 or 22.In other specific embodiments, described cupredoxin is the Laz albumen from Neisseria meningitidis or Diplococcus gonorrhoeae.

The invention provides the aminoacid sequence variant of cupredoxin or cytopigment, it is compared with wild type peptide has amino acid alternative, that lack or insert.Variant of the present invention can be the clipped form of wild type peptide.In some embodiments, described composition comprises peptide, and described peptide is made up of the district that is less than the total length wild type peptide of cupredoxin or cytopigment.In some embodiments, described composition comprises peptide, and described peptide is surpassed about 15 residues or surpasses about 20 residues and formed by about 10 residues that surpass of the cupredoxin of brachymemma or cytopigment.In some embodiments, described composition comprises peptide, and described peptide is no more than about 50 residues by about 100 residues that are no more than of the cupredoxin of brachymemma or cytopigment, is no more than about 40 amino or is no more than about 30 residues to form.In some embodiments, composition comprises peptide, described peptide and cupredoxin or cytopigment, more particularly has aminoacid sequence homogeny, the aminoacid sequence homogeny at least about 95% or at least about 99% aminoacid sequence homogeny at least about 90% with SEQ ID NO:1-20 or 22.

In specific embodiment, the variant of cupredoxin comprises Pseudomonas aeruginosa azurin residue 36-89.In other embodiments, the variant of described cupredoxin is made up of Pseudomonas aeruginosa azurin residue 36-89.In other specific embodiments, described variant is made up of the residue of equal value of the cupredoxin that is different from azurin.

Consideration can design with azurin residue 36-89 has similar active other cupredoxin variants.For this reason, use BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) that target cupredoxin aminoacid sequence and Pseudomonas aeruginosa azurin sequence are carried out parallelism, thereby evaluation is positioned at the residue of equal value that exists on relevant residue on the Pseudomonas aeruginosa azurin aminoacid sequence, the target cupredoxin sequence and the residue of equal value of cupredoxin.

Described variant also comprises the peptide made from the synthesizing amino acid of non-natural existence.For example, the amino acid that non-natural exists can be incorporated in the variant peptides to prolong or to optimize the transformation period of described composition in blood flow.These variants include but not limited to D, L-peptide (diastereomer) (Futakiet al., J.Biol.Chem.276 (8): 5836-40 (2001); Papo et al., Cancer Res.64 (16): 5779-86 (2004); Miller et al., Biochem.Pharmacol.36 (1): 169-76, (1987)); Peptide (the Lee et al. that contains rare amino acid, J.Pept.Res.63 (2): 69-84 (2004)), and mix non-natural amino acid hydrocarbon polymer nail pin (hydrocarbon stapling) (Schafmeister et al., the J.Am.Chem.Soc.122:5891-5892 (2000) then that contains alkene; Walenski et al., Science 305:1466-1470 (2004)) and the peptide that comprises ε-(3,5-phenodiazine benzoyl)-Lys residue.

In other embodiments, peptide of the present invention is the derivative of cupredoxin or cytopigment.The derivative of described cupredoxin or cytopigment is chemically modified forms of described peptide, makes described peptide still keep some primary activities.For example " derivative " of azurin can be the azurin of chemically modifying, and it has kept it and has suppressed the ability of plasmodium mass formed by blood stasis in the mammalian cell.Interested chemically modified includes but not limited to amidation, acetylize, sulfation, polyoxyethylene glycol (PEG) modification, phosphorylation and the glycosylation of peptide.In addition, the deutero-peptide can be the fusion of cupredoxin or cytopigment or its variant, derivative or structural equivalents and compound, and described compound is such as but not limited to another kind of peptide, drug molecule or other treatment agent or medicine or detectable probe.Interested derivative comprises the chemically modified form, transformation period in blood flow can be extended or optimize by its peptide of the present invention and composition, described chemically modified form is for example by well known to a person skilled in the art that several method includes but not limited to peptide (the Monk et al. of cyclisation, BioDrugs 19 (4): 261-78, (2005); DeFreest et al., J.Pept.Res.63 (5): 409-19 (2004)), end modified (the Labrie et al. of N-and C-, Clin.Invest.Med.13 (5): 275-8, (1990)), and mix non-natural amino acid hydrocarbon polymer nail pin (Schafmeister et al., the J.Am.Chem.Soc.122:5891-5892 (2000) then that contains alkene; Walenski et al., Science 305:1466-1470 (2004)) finish.

In one embodiment, with described cupredoxin or cytopigment, or its variant, derivative or structural equivalents are merged to the H.8 district from the Laz of Neisseria meningitidis or Diplococcus gonorrhoeae.An example of these peptides is H.8-Paz fusion roteins of describing in embodiment 4.In specific embodiment, H.8 merged to cupredoxin or cytopigment, or the C-end of its variant, derivative or structural equivalents.In another specific embodiment, described H.8 district is SEQ ID NO:21, or its variant, derivative or structural equivalents.

The peptide of considering composition of the present invention can be cupredoxin or cytopigment more than a kind of variant, derivative and structural equivalents.For example, described peptide can be by the clipped form of the azurin of PEGization, thus make its be variant be again derivative.In one embodiment, that use contains band alkene is the α of thing, and α-dibasic alpha-non-natural amino acid forms total hydrocarbon " bail " by the catalytic olefin metathesis of ruthenium then and synthesizes peptide of the present invention.(Scharmeister et al.，J.Am.Chem.Soc.122：5891-5892(2000)；Walenskyet al.，Science 305：1466-1470(2004))。In addition, can merge with other peptides as the peptide of the structural equivalents of azurin, thus produce be structural equivalents be again the peptide of derivative.These examples only are exemplary rather than restriction the present invention.The variant of cupredoxin or cytopigment, derivative or structural equivalents can in conjunction with or can debond copper.

In another embodiment, described peptide can be the structural equivalents of cupredoxin or cytopigment.The example of determining the research of the remarkable structural homology between cupredoxin and cytopigment and other albumen comprises Toth et al. (Developmental Cell 1:82-92 (2001)).Especially, by using the VAST algorithm to determine remarkable structural homology (Gibrat et al., Curr Opin Struct Biol6:377-385 (1996) between cupredoxin or cytopigment and its structural equivalents; Madej et al.Proteins 23:356-3690 (1995)).In specific embodiment, be less than about 10 from the texture ratio VAST p value of cupredoxin or cytopigment and its structural equivalents ^-3, less than about 10 ^-5, or less than about 10 ^-7In other embodiments, the remarkable structural homology between cupredoxin or cytopigment and its structural equivalents is by using DALI algorithm (Holm ﹠amp; Sander, J.Mol.Biol.233:123-138 (1993)) determine.In specific embodiment, the DALIZ value of pairing structure comparison is at least about 3.5, at least about 7.0, or at least about 10.0.

In another embodiment, the Fab fragment of the variant of cupredoxin or derivative and G17.12 mouse monoclonal antibody has significant structural homology.Example how to determine this structural similarity can be referring to embodiment 3.Especially, the remarkable structural homology between the Fab fragment of cupredoxin and G17.12 mouse monoclonal antibody can use the VAST algorithm to determine (Gibrat et al., id.; Madej et al., the same).In specific embodiment, can be from the segmental texture ratio of the Fab VASTp value of cupredoxin and G17.12 mouse monoclonal antibody less than about 10 ^-4, less than about 10 ^-5, less than 10 ^-6Or less than about 10 ^-7In other specific embodiments, the segmental texture ratio of the Fab VAST value of cupredoxin and G17.12 mouse monoclonal antibody can be greater than about 9, greater than about 10, greater than about 11 or greater than about 12.

In some embodiments, its variant, derivative or structural equivalents have Pseudomonas aeruginosa azurin, Pseudomonas aeruginosa cytochrome c ₅₅₁, human cytochrome c or cyanobacteria cytopigment f some functional characters.In specific embodiment, the parasitemia of the malaria in the red corpuscle of peptide inhibition malaria infection of the present invention, more particularly, the parasitemia of the plasmodium falciparum in the HRBC of inhibition falciparum infection.The present invention also provides cupredoxin and cytochrome c ₅₅₁Variant, derivative and structural equivalents, the parasitemia that it has kept in the red corpuscle that suppresses malaria infection more particularly suppresses the ability of the parasitemia of plasmodium falciparum in the HRBC of falciparum infection.The inhibition of the parasitemia of plasmodium falciparum can be determined by the method for describing among the embodiment 6 in the HRBC of falciparum infection.

Because it is now know that cupredoxin and cytopigment can suppress the parasitemia in the red corpuscle of malaria infection, therefore may design variant, derivative and the structural equivalents that keeps active cupredoxin of this anti-malarial and cytopigment now.For example can be by producing the various variants and the derivative " storehouse " of cupredoxin and cytopigment, use a kind of in many methods known in the art then, for example illustrative method is tested every kind anti-malarial activity among the embodiment 6, to produce these variants and derivative.Consider that variant, derivative and the structural equivalents with the active cupredoxin of anti-malarial and cytopigment produced can replace or be used from the method for the present invention with cupredoxin and cytopigment one that this paper mentions.The method of this selection variant and derivative can be suitable for Pseudomonas aeruginosa azurin disclosed herein, Pseudomonas aeruginosa cytochrome c ₅₅₁, human cytochrome c or cyanobacteria cytopigment f any activity.

In other embodiments, peptide of the present invention suppresses plasmodial time multiplexed cell system in the HRBC.The method of measuring plasmodial time multiplexed cell system is well known in the art, and a kind of such method is described in embodiment 2.

In some embodiments, peptide of the present invention with on the statistics greater than the RA of non-binding reference protein in conjunction with PfMSP1-19 and/or PfMSP1-42 plasmodium falciparum surface proteins.By this activity of using as the surface plasma resonance analysis of description among the embodiment 5 can test peptides.Whether in conjunction with alternative additive method be well known in the art, also can use if measuring a kind of albumen.

In another embodiment, peptide of the present invention with on the statistics greater than the RA of non-binding reference protein in conjunction with ICAM-3 or NCAM.By this activity of using as the surface plasma resonance analysis of description in embodiment 7 and 5 can test peptides.Whether in conjunction with alternative additive method be well known in the art, also can use if measuring a kind of albumen.

In some specific embodiments, inducing peptide mammalian cancer cells of the present invention, the more particularly apoptosis in the J774 cell.The ability of inducing peptide apoptosis can be by using MITOSENSOR ^TMAPOLERT ^TMMitochondrial Membrane Sensor test kit (Clontech Laboratories, Inc., Palo Alto, California U.S.A.) uses mitosensorApoAlert ^TMConfocal microscopy, by using the method for describing among the Zou et al. (J.Biol.Chem.21 A:11549-11556 (1999)) to measure caspase-8, caspase-9 and caspase-3 is active and by using for example APOLERT ^TMThe dna fragmentation test kit (Clontech Laboratories, Inc., Palo Alto, California, U.S.A.) the nuclear dna fragmentation of detection apoptosis induction is observed.

In another specific embodiment, inducing peptide mammalian cancer cells of the present invention, more particularly the cell cessation of growth cessation in the J774 cell.The cell cessation of growth cessation can for example be measured by the method among the Yamada et al. (PNAS101:4770-4775 (2004)) by measuring the inhibition degree of cell cycle progress.In another specific embodiment, peptide of the present invention suppresses mammalian cancer cells, more particularly the progress of the cell cycle in the J774 cell.

Cupredoxin

These little blue copper proteins (cupredoxin) are to participate in the electron transfer protein (10-20kDa) of bacterium electron transport chain or the albumen of unknown function.Cupric ion is by albumen substrate combination individually.The planar delta of the special distortion of two Histidines of copper and a halfcystine part arranged produced the very special electronic property and the intensive blueness in metal site.In under high resolving power, characterized a large amount of cupredoxins from crystallography.

Described cupredoxin generally has low sequence homology, but has high structural homology.(Gough & Clothia，Structure 12：917-925(2004)；De Rienzo et al.，Protein Science 9：1439-1454(2000).)。For example, the aminoacid sequence of azurin deflects plain B 31% homogeny is arranged with orange is green, with iron sulphur bacterium azurin 16.3% homogeny is arranged, and 20.3% homogeny is arranged and with false azurin 17.3% homogeny is arranged with plastocyanin.See Table 1.Yet these proteic structural similarities are more significant.Azurin is 10 with the green texture ratio VAST p value that deflects plain B of orange ^-7.4, azurin and iron sulphur bacterium azurin be 10 ^-5, azurin and plastocyanin be 10 ^-5.6, and azurin and false azurin is 10 ^-4.1

All cupredoxins all have Greece's key (Greek key) β-tubbiness or β-sandwich the folding of eight chains, and have the site structure of high conservative.(De Rienzo et al.，ProteinScience 9：1439-1454(2000).)。Around azurin, cyanin, cyanobacteria plastocyanin, the proteic copper of cucumber alkalescence site, exist because of the many long-chain fats family residue remarkable hydrophobicity sticking patch (patch) of methionine(Met) and leucic existence for example, and around the plastocyanin of false azurin and eucaryon, have the hydrophobicity sticking patch of less degree.The same.The hydrophobicity sticking patch is found on also less degree ground in star cyanin and iron sulphur bacterium azurin copper site, but takes on a different character.The same.

Table 1: the azurin from Pseudomonas aeruginosa (1JZG) and other proteic sequences and structure parallelism that use the VAST algorithm.

PDB	Parallelism length 1	Amino acid homogeny %	The P value 2	Score value 3	RMSD 4	Describe
							1AOZA2 1QHQ_A 1V54B 1 1GY2A 3MSPA	82 113 79 92 74	18.3 31 20.3 16.3 8.1	10e-7 10e-7.4 10e-6.0 10e-5.0 10e-6.7	12.2 12.1 11.2 11.1 10.9	1.9 1.9 2.1 1.8 2.5	Vitamin C oxidase orange is green to deflect the plain B cytochrome c oxidase iron sulphur bacterium azurin main sperm protein that moves 5

1IUZ 1KGYE 1PMY

74 90 75

20.3 5.6 17.3

10e-5.6 10e-4.6 10e-4.1

10.3 10.1 9.8

2.3 3.4 2.3

The false azurin of plastocyanin Ephrinb2

¹Parallelism length: the right number of C-alpha atom of equal value that overlaps between two structures, promptly how many residues have been used to calculate the 3D coincidence.

²P-VAL:VAST p value is the measuring of significance of comparison, is expressed as probability.For example, if the p value is 0.001, then pure chance sees that the paired probability of this quality is 1000: 1.To the effect of multiple comparisons, using this hypothesis is the p value that 500 kinds of independences in the MMDB database and irrelevant structural domain type are adjusted VAST.Therefore, shown p value is corresponding to the p value divided by the right paired comparison of each structural domain of 500.

³Score value: VAST structural similarity score value.The number of secondary structure element of this numeral and coincidence is relevant with the quality of coincidence.Higher VAST score value is with higher similar relevant.

⁴RMSD: unit is that the rootmean-square of dust overlaps remainder.After the best of two structures overlaps, this number is calculated the square root of making the mean square distance between the C-alpha atom of equal value.Notice that the degree of RMSD value and structure parallelism is proportional, and when using RMSD, essentially consider this size as the descriptor of overall structure similarity.

⁵The main sperm protein of beautiful nematode (C.elegans) is proved to be the ephrin antagonist (Kuwabara in the oocyte maturation, 2003 " The multifaceted C.elegans majorsperm protein:an ephrin signalling antagonist in oocyte maturation " Genes and Development, 17:155-161.

Azurin

Azurin is the cuproprotein that contains of 128 amino-acid residues, and it belongs to the cupredoxin family of electron transport in involved in plant and some bacterium.Azurin comprises the azurin from Pseudomonas aeruginosa (PA) (SEQ ID NO:1), Achromobacter xylosoxidans and alcaligenes dentrificans (A.denitriflcans) (SEQ ID NO:8).(Murphy et al.，J.Mol.Biol.315：859-871(2002))。Aminoacid sequence homogeny between the azurin changes between 60-90%, and these albumen have shown strong structural homology.All azurins all have the characteristic p-sandwich structure of band Greece key motif, and single copper atom always is positioned at proteic same area.In addition, azurin has the hydrophobicity of the neutral basically sticking patch around the copper site.The same.

Plastocyanin

Plastocyanin is the soluble proteins of cyanobacteria, algae and plant, and its each molecule contains a part copper and its oxidised form is blue.They are present in the chloroplast(id), and wherein they work as electron carrier.Since the structure of comfortable definite white poplar plastocyanin in 1978, determined the structure of algae (scenedesmus obliquus (Scenedesmua), Enteromorpha (Enteromorpha), chlamydomonas (Chlamydomonas)) and plant (French bean) plastocyanin by crystallography or NMR method, the structure of white poplar has been fine to 1.33 dust resolving power.SEQ ID NO:4 shows the aminoacid sequence from the plastocyanin of thermophilic cyanobacteria bedding seat algae.

Although there is sequence difference (62% sequence homogeny is for example arranged) between the plastocyanin of algae and vascular plant between clothing Monas (Chlamydomonas) and white poplar albumen, three-dimensional structure is (for example the C alpha position between Chlamydomonas and white poplar albumen has the root-mean-square deviation of 0.76 dust) of guarding.Constitutional features is included in tetrahedron copper binding site, significantly the negative film section and the smooth water repellent surface of distortion of an end of the antiparallel β of eight chains-bucket.Optimised for its transfer transport function copper site, think that negativity, hydrophobicity sticking patch have participated in the identification of physiological response mating partner.Negativity that chemically modified, the experiment of crosslinked and site-directed mutagenesis are verified, hydrophobicity sticking patch with the binding interactions of cytopigment f in importance, and confirmed to relate to the model of significant electron transfer on two functions of plastocyanin.An electron transfer of inferring is short relatively (about 4 dusts), relate to the copper part His-87 that solvent exposes in the hydrophobicity sticking patch, and another is than long (about 12-15 dust), relate to the nearly conserved residues Tyr-83 in the negativity fragment, Redinbo et al., J.Bioenerg.Biomembr.26:49-66 (1994).

Iron sulphur bacterium azurin

Iron sulphur bacterium azurin is the single chain polypeptide that contains blue copper that obtains from thiobacillus (being called sour sulphur rod bacterium (Acidithiobacillus) now).Determined x-ray crystal structure by the unusual diffraction of multi-wavelength, and be fine to the resolving power of 1.9 dusts from the oxidised form of the very stable and highly oxidized cupredoxin iron sulphur bacterium azurin (SEQ ID NO:5) of thiobacillus ferrooxidant.Iron sulphur bacterium azurin is made of core β-sandwich folding, and described core β-sandwich folding is made up of the β-lamella of six chains and seven chains.The same with other cupredoxins, that cupric ion is arranged by the distortion tetrahedron by cluster four conserved residues (His85, Cys138, His143, Met148) coordination.Walter，R.L.et al.，J.Mol.Biol.，vol.263，pp-730-51(1996)。

False azurin

False azurin is the single chain polypeptide family that contains blue copper.From the aminoacid sequence of the false azurin of driffractive ring achromobacter shown in the SEQ ID NO:6.The x ray structure analysis of false azurin shows that it and azurin have similar structure, but has low sequence homology between these albumen.There are two main differences between the one-piece construction of false azurin and azurin.With respect to azurin, in false azurin, exist the C-terminal that constitutes by two α spirals to extend.In the intermediate peptide district, azurin contains the ring of extension, is shortened in false azurin, and its formation contains the limb (flap) of short alpha-helix.Unique main difference in copper atom site is the conformation and the Met-S copper key length of MET side chain, and its ratio in false azurin is significantly little in azurin.

Plant cyanin (Phytocyanin)

The albumen that can be accredited as the plant cyanin includes but not limited to the blue copper protein of inferring in cucumber basic protein, star cyanin, mavicyanin, umecyanin, peel of Fructus Cucumidis sativi cupredoxin, the pea pod and from the blue copper protein of Arabidopis thaliana (Arabidopsis thaliana).In all albumen except that cucumber basic protein and pea pod albumen, the axle methionine(Met) part that is usually located at blue copper site is substituted by glutamine.

The green element that deflects of orange

From thermophilic green slide photosynthetic bacterium orange green subdue separate the bacterium three kinds be called the orange green deflect plain A, the orange green deflect plain B-1 and the orange the green little blue copper protein that deflects plain B-2.Two B forms are glycoprotein, and it has character much at one each other, but are different from the A form.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that apparent monomer molecule amount is 14 (A), 18 (B-2) and 22 (B-1) kDa.

Determine the orange green aminoacid sequence that deflects plain A, and proved the orange green polypeptide that plain A is 139 residues that deflects.(Van Dreissche et al.，Protein Science8：947-957(1999).)。If they are metal ligands of evolution conservative in known little cuproprotein plastocyanin and the azurin, then His58, Cys123, His128 and Met132 are by way of expectations at interval.Secondary structure prediction also shows, orange is green to deflect element and have and be similar to from the azurin of Pseudomonas aeruginosa with from the general beta-barrel structure of the plastocyanin of Leaf of David Poplar.Yet orange is green to deflect the sequence signature that element seems to have two kinds little cuproprotein sequence kinds.Roughly the overall similarity with the blue plain consensus sequence of homoplasmon is identical with the overall similarity of the consensus sequence of azurin, and promptly 30.5%.The green N-end sequence district 1-18 that deflects element of orange significantly is rich in glycine and hydroxy-amino-acid.The same.Referring to from the orange green orange green exemplary aminoacid sequence SEQ ID NO:16 (NCBI albumen database numbering No.AAM12874) that deflects plain A chain that subdues bacterium.

Orange is green to deflect plain B molecule to have a cupredoxin of standard folding.After deliberation from the orange green orange green crystalline structure that deflects plain B of subduing bacterium.(Bond et al.，J.Mot Biol.306：47-67(2001).)。Except that the terminal chain of other N-, the structure of this molecule and bacterium cupredoxin, azurin is closely similar.As in other cupredoxins, a Cu part is on the chain 4 of polypeptide, and other three parts are on the big ring between chain 7 and 8.With reference to the amino acid between back three parts the geometry in Cu site is discussed at interval.The orange green Cu binding domains that deflects plain B that crystallography characterizes may be tied to the endochylema side of cytoplasmic membrane by the terminal afterbody of N-, and the terminal afterbody performance of described N-is known in other the film associated electrical transferrin with some to be thing (tether) sequence homogeny significantly.The aminoacid sequence of B form is described in McManus et al. (J.Biol Chem.267:6531-6540 (1992)).Referring to from the orange green orange green exemplary aminoacid sequence SEQ IDNO:17 (NCBI albumen database numbering No.1QHQA) that deflects plain B chain that subdues bacterium.

The star cyanin

The star cyanin is the subclass of the omnipresence family plant cyanin of plant cupredoxin.Exemplary sequence this paper of star cyanin comprises SEQ ID NO:15.From the star cyanin beans azurin (Koch et al., J.Am.Chem.Soc.127:158-166 (2005)) of horseradish and the crystalline structure of cucumber star cyanin (Hart el al, Protein Science 5:2175-2183 (1996)).This albumen has all similar to the other plant cyanin and folds.Ephrin B2 albumen ectodomain tertiary structure has and the significant similarity of star cyanin.(Toth et al.，Developmental Cell 1：83-92(2001).)。The exemplary aminoacid sequence of star cyanin is referring to the numbering No.1JER in the state-run biotechnology information center albumen database, SEQID NO:15.

The cucumber basic protein

Proteic exemplary aminoacid sequence this paper comprises SEQ ID NO:18 from cucumber alkalescence.For the crystalline structure of the cucumber basic protein (CBP) of I type blue copper protein has been fine to 1.8 dust resolving power.This molecule is similar to other blue copper proteins, has Greece's key beta-barrel structure, and just bucket is called " β-sandwich " or " β-volume " better at a side opening.(Guss et al.，J.MolBiol.262：686-705(1996).)。EphrinB2 albumen ectodomain tertiary structure and cucumber basic protein have the similarity (root-mean-square deviation 1.5 dusts of 50 α carbon) of height.(Toth etal.，Developmental Cell 1：83-92(2001).)。

The Cu atom has common blue copper NNSS coordination altogether, bond distance Cu-N (His39)=1.93 dust, Cu-S (Cys79)=2.16 dust, Cu-N (His84)=1.95 dust, Cu-S (Met89)=2.61 dust.Disulfide linkage (Cys52)-S-S-(Cys85) seems to play an important role in the stable molecule structure.Polypeptide is folding to be that the typical case of blue copper protein subfamily (plant cyanin) and nonmetal protein artemisiifolia anaphylactogen Ra3 institute has, and artemisiifolia anaphylactogen Ra3 and CBP have sequence homogeny highly.The albumen that is accredited as the plant cyanin at present is the blue copper protein of inferring in CBP, star cyanin, mavicyanin, umecyanin, peel of Fructus Cucumidis sativi cupredoxin, the pea pod and from the blue copper protein of Arabidopis thaliana.In all albumen except CBP and pea pod albumen, the axle methionine(Met) part that is present in blue copper site is usually substituted by glutamine.The proteic exemplary sequence of cucumber alkalescence is seen NCBI albumen database numbering No.2CBP, SEQ ID NO:18.

Cytopigment

Cytochrome C ₅₅₁

Cytochrome C from Pseudomonas aeruginosa ₅₅₁(Pa-C ₅₅₁) be the monomer redox protein (SEQ ID NO:2) of 82 amino-acid residues, its physiology electron donor as nitrite reductase participates in the alienation denitrification.Pa-C ₅₅₁Functional property studied widely.Provide test with unphysiologic little inorganic oxide reducing agents and with the reaction of the cytochrome c of other macromole such as blue copper protein, eucaryon and physiology mating partner nitrite reductase to the protein-protein transfer transport.

Pa-C as the member of bacterium I cytochromoid ₅₅₁Three-dimensional structure shown that to have single low spin protoheme and typical polypeptide that His-Met connects folding, but it stays protoheme pyrrole ring II and III edge (Cutruzzola et al., the J.Inorgan.Chem.88:353-61 (2002)) of exposure.The shortage of the 20 residue ω ring that exists in the Mammals I cytochromoid causes the further exposure at protoheme edge under the propionic salt level.Pa-C ₅₅₁It is very anisotropic that the charged residue on surface distributes: a side is rich in acidic residues, and opposite side has the ring of the positive side chain that mainly is Methionin, and it is positioned at around the border of the hydrophobicity sticking patch in protoheme crack.This fragment comprises residue Gly11, Val13, Ala14, Met22, Val23, Pro58, Ile59, Pro60, Pro62, Pro63 and Ala65.Anisotropic charge distribution produces big moment of dipole, and it forms for charge-transfer complex is important.

More than to Pa-C ₅₅₁The charge distribution of describing is reported in other electron transfer proteins and their electron acceptor(EA).In addition, the site-directed mutagenesis of the residue in hydrophobicity or the charged fragments is modified and has been shown that superficial complementarity is for the importance of combination and transfer transport for different albumen.For example.The hydrophobicity sticking patch for from the evidence of the dependency of the transfer transport character of the azurin of Pseudomonas aeruginosa from residue Met44 and Met64 being changed into the research that positively charged and the amino acid whose mutant of negative electricity carry out.The same.

Cytochrome c type structural domain has by folding that a series of α spirals that are used for surrounding covalently bound protoheme in the hydrophobicity pocket and reverse corner constitute.This structural domain can be at single structure territory cytochrome c albumen for example cytochrome c 6, cytochrome c ₅₅₂, cytochrome C ₄₅₉With find among the mitochondrial cytochrome c.Cytochrome c type structural domain is present in many other albumen, for example in Cytochrome cd 1-nitrite reductase as the terminal heme c structural domain of N-, in the quinoprotein ethanol dehydrogenase as C-end structure territory, in quinone reduced hematin amine dehydrogenase A chain as structural domain 1 and 2, and at cytochrome b c ₁In the mixture as cytochrome b c ₁Structural domain.The structural analysis of VAST algorithm is (from the cytochrome c of Pseudomonas aeruginosa ₅₅₁As the inquiry thing) only show to have significant structure by the pair cell pigment contiguous (the P value is 10 ^-10.3To 10 ^-4.5Between).

Using method

The invention provides the method that treatment has malaria infection or has the patient who obtains infection risk, or suppress the method for plasmodial propagation.These methods comprise cupredoxin or cytopigment or its variant, derivative or the structural equivalents to the parasitemia of the mammalian cell of patient or insect vector administration inhibition malaria infection.The inhibition of parasitemia can be measured by many methods well known in the art.In embodiment 6, describe a kind of method, measured the inhibition of parasitemia in the HRBC of malaria infection.In other embodiments, described cupredoxin or cytopigment or its variant, derivative or structural equivalents suppress plasmodial time multiplexed cell system in the HRBC, and it is delivered medicine to patient or insect vector.The method of measuring plasmodial time multiplexed cell system is well known in the art, and a kind of such method is described in embodiment 2.Mode of the present invention is not limited to any specific mechanism, the inhibition of parasitemia can cause by many factors, parasiticly in the hemocyte that described factor includes but not limited to suppress to infect duplicates, suppresses hemocyte, the inhibition that suppresses the parasite growth cycle that parasitic infection do not infect and suppress parasite to enter mammalian cell.

The invention provides the method that suffers the patient of plasmodium infection by at least a protein for treatment of effective dosage, described albumen is cupredoxin or cytopigment or its variant, derivative or structural equivalents.Can be any Mammals, particularly people patient that possible be infected by plasmodium by the patient of this method treatment.The mammiferous plasmodium of known infection includes but not limited to plasmodium falciparum, Plasmodium vivax, Bai Shi plasmodium (P.berghei) (rodent is specific), Plasmodium yoelii (P.yoelli) (mouse is specific), cynomolgus monkey plasmodium (P.cynomolgi) and Plasmodium knowlesi (P.knowlesi) (monkey is specific).

Also known, cupredoxin and cytochrome c ₅₅₁It also is effective infecting at HIV-1, as common application " the Compositions And Methods For Treating HIVInfection With Cupredoxin And Cytochrome c " U.S. Provisional Patent Application of submitting to _ _ in disclosed, its disclosure is incorporated herein by reference clearly.In addition, the coinfection of HIV and malaria is in many zones in the world, and particularly inferior the Sahara Africa right and wrong are usually seen.In some embodiments, the patient who infected by plasmodium is infected by HIV also.In some embodiments, methods of treatment of the present invention also comprises the anti-HIV medicine of administration.In some embodiments, will resist HIV medicine Combined Preparation.

The present invention also provides the doubtful method that has contacted plasmodial patient of at least a peptide treatment by effective dosage, and described peptide is variant, derivative or the structural equivalents of cupredoxin and/or cytopigment or cupredoxin or cytopigment.For example, if the patient lives in or other patient's that traveled malaria infection is common world region, then the patient can doubtfully contact with plasmodium.The treatment of this method can begin when the patient will or contact with plasmodium.With plasmodial contact the most common by with insect vector for example mosquito contact generation, thereby think that these insects and the abundant zone of plasmodium belong to the patient and will have high probability to contact plasmodial zone.These zones include but not limited to the part of Africa, Asia and Latin America in the world.In addition, if the patient contacts with the blood that is infected by plasmodium, or be exposed to plasmodium wittingly, or injected blood or the medicine that is polluted by parasite by accident, then the patient can doubtfully contact with plasmodium.

The variant of cupredoxin or cytopigment or cupredoxin or cytopigment, derivative or structural equivalents can deliver medicine to the patient by well known to a person skilled in the art many approach and many modes.In specific embodiment, the variant of described cupredoxin or cytopigment or cupredoxin or cytopigment, derivative or structural equivalents oral administration, part, by sucking, pass through drug administration by injection, more specifically, intravenously, intramuscular or subcutaneous administration.

In one embodiment, described method can comprise to the composition that comprises cupredoxin or cytopigment or its variant, derivative or structural equivalents of a unitary dose of patient's Combined Preparation and the composition that comprises antimalarial drug and/or anti-HIV medicine of a unitary dose, with any order.These compositions can be in approximately identical time or administration in the about preset time of administration after another, for example, and after another about 1 minute to about 60 minutes, or about 1 hour to about 12 hours.

The present invention also provides by at least a in variant, derivative or the structural equivalents of the insect vector administration cupredoxin in described colony or cytopigment or cupredoxin or cytopigment and suppresses to carry the method that plasmodium is propagated in the plasmodial insect vector colony, and its amount is for effectively reducing the communicable amount of parasite in the coexistence Mammals colony.In specific embodiment, described insect vector is a mosquito, more particularly the mosquito that belongs to from malarial mosquito (Anophelesgambiae).In the method, the administration of the variant of cupredoxin or cytopigment or cupredoxin or cytopigment, derivative or structural equivalents can be attended by the composition that places described insect vector to consume described peptide, but any way that described peptide is contacted with plasmodial enteron aisle in the insect vector all is considered.Many methods to the insect colony administration chemical substance that produces this consumption are known in the art.

In another embodiment, the genetic elements propagated that passes to another from a mosquito will be operably connected to the cupredoxin encoding sequence that is operably connected with constitutive promoter, the variant of cupredoxin or cytopigment or cupredoxin or cytopigment, derivative or structural equivalents will produce in by the malarial mosquito of falciparum infection, and will disturb plasmodium falciparum duplicating in mosquito/survive.

Include but not limited to variant, derivative or the structural equivalents gene fusion of cupredoxin or cytopigment or cupredoxin or cytopigment on to other modes of the described peptide of insect vector administration from other proteic genes of being consumed by described insect usually.The amount that delivers medicine to the peptide of insect vector should be effectively to reduce the communicable amount of plasmodium in the Mammals when described insect vector contacts with Mammals.In specific embodiment, dosage should be effectively to reduce the communicable amount of plasmodium when described insect vector contacts with the people.

Mosquito larvae is applicable to the present invention, and preferably, employed promotor is a strong promoter.The promotor that can obtain two kinds of replaceabilities is for using: derivable and constitutive promoter.Inducible promoter comprises for example heat-shocked promotor.Preferably, described heat-shocked promotor is an insect heat-shocked promotor, drosophila melanogaster (Drosophila melanogaster) hsp70 promotor for example, and it can drive the expression of gene in comprising the allos organism of medfly.Use medfly hsp70 promotor (Papadimitriou et al., InsectMol Biol 7:279-90 (1998)) is also contained in the present invention.Selectable system can be based on using antibiotic tetracycline to induce.(Heinrich and Scott，PNAS 97：8229-8232，(2000))。

The heat-shocked promotor can be induced by the temperature that improves the medfly culture condition.For example, under 23-25 ℃, the hsp70 promotor is low-level active or do not have an activity at all.This allows the insect larvae growth, stress not and do not generate institute's inductive by heterologous protein.Yet under high temperature, for example under 37-42 ℃, the hsp70 promotor is induced and with the high level expression heterologous protein.

But can make up inducible promoters according to known induced gene controlling elements.For example, can be by the element structure inducible promoters of combination to medicine or hormone response, described medicine or hormone can administrations in meals.In preferred embodiments, human estrin response element (ERE) can be used to regulate the expression of target protein, and condition is second encoding sequence conversion insect with the expressing human estrogen receptor.

Constitutive promoter also can be used for expressing this albumen and/or other albumen that insect larvae needs.For example, constitutive promoter can be the tenuigenin actin promoter.Cloned drosophila melanogaster tenuigenin actin promoter (Act5C), and be highly active (Huynh and Zieler, J.Mol.Biol.288:13-20 (1999)) in mosquito.Tenuigenin actin gene and their promotor also can be separated from other insects that comprise medfly.Other examples comprise the cytotubule protein promoter, for example medfly cytotubule protein promoter.

Can use the promotor of control secrete polypeptide, choose wantonly and use, to instruct the secretion of albumen to hemolymph with the appropriate signal sequence.For example, (Benes et al., Mol.Gen.Genet 249 (5): 545-556 (1995)) can to adopt the serum protein promotor of larva.

The large-scale breeding technology of mosquito is by high level of development.For protein production, the larva culture that starts at different time can come synchronously by suitable temperature variation scheme.Because the speed of growth depends on the temperature of environment, so this is possible; The speed of growth 18 ℃ of following larvas reduces about 50%.

The pharmaceutical composition that comprises cupredoxin and/or cytopigment and its variant and derivative

The pharmaceutical composition that comprises cupredoxin or cytopigment or its variant, derivative or structural equivalents can for example prepare by conventional mixing, dissolving, granulation, system ingot, emulsification, packing, embedding (entrapping) or freeze drying process in the mode of any routine.Basically pure cupredoxin and/or cytopigment and its variant, derivative and structural equivalents can easily make up with pharmaceutically acceptable carrier well known in the art.These carriers make goods be configured to tablet, pill, lozenge, capsule, liquid agent, gelifying agent, syrup, slurries, suspensoid etc.The carrier or the vehicle that are fit to can also comprise for example weighting agent and cellulosics.Other vehicle can comprise for example seasonings, tinting material, release agent, thickening material and other acceptable additive, auxiliary agent or tackiness agent.In some embodiments, described pharmaceutical preparation is substantially free of sanitas.In other embodiments, described pharmaceutical preparation can contain at least a sanitas.The general approach of pharmaceutical dosage form is referring to Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems (Lippencott Williams ﹠amp; Wilkins, Baltimore MD (1999)).

The composition that comprises cupredoxin or cytopigment or its variant, derivative or structural equivalents that uses among the present invention can comprise in every way by injection (for example intracutaneous, subcutaneous, intramuscular, intraperitoneal etc.), by suck, by topical, by suppository, by using through the skin patch or passing through oral administration.Can be about the general information of drug delivery system referring to Ansel et al., the same.In some embodiments, can prepare the composition that comprises cupredoxin or cytopigment or its variant, derivative or structural equivalents and also directly use, use especially for subcutaneous and intravenous injection as injection.Especially, injectable preparation can be used for the treatment of valuably and have the malaria infection risk, has malaria infection or the patient of malaria infection has been arranged.The composition that comprises cupredoxin or cytopigment or its variant, derivative or structural equivalents also can be in that for example polypropylene glycol or similar Drug coating mix the back oral administration with protective material.

When the drug administration by injection, cupredoxin or cytopigment or its variant, derivative or structural equivalents can be formulated in the aqueous solution, particularly in the physiology compatible buffers for example in Hanks liquid, Ringer's solution or the normal saline buffer solution.This solution can comprise preparaton for example suspending agent, stablizer and/or dispersion agent.Perhaps, cupredoxin or cytopigment or its variant, derivative or structural equivalents can be that the powder form is used for before use with for example aseptic apirogen water preparation of the vehicle that is fit to.In some embodiments, described pharmaceutical composition does not contain immunoreactive adjuvant or any other material that is added with the stimulation of enhancing peptide.In some embodiments, described pharmaceutical composition comprises the immunoreactive material of inhibition to peptide.

When by the intravenous fluid administration, the intravenous fluid that is used for described cupredoxin of administration or cytopigment or its variant, derivative or structural equivalents can be made up of crystal or colloid.Crystal used herein is the aqueous solution of inorganic salt or other water soluble molecules.Colloid used herein contains for example gelatin of big insoluble molecule.Intravenous fluid may be aseptic.

The crystal liquid that can be used for intravenous administration includes but not limited to 5% glucose solution (being sometimes referred to as D5W) of physiological saline (concentration is 0.9% sodium chloride solution), woods lattice lactate buffer solution or Ringer's solution and Yu Shuizhong, described in table 2.

The composition of the crystalloid solution that table 2. is common

Solution	Other titles	[Na+]	[Cl-]	[glucose]
					D5W 2/3﹠1/3 manages salt solution physiological saline woods lattice lactate buffer solution half a lifetime *	5% glucose, 3.3% glucose/0.3% salt solution 0.45%NaCl 0.9%NaCl Ringer's solution	0 51 77 154 130	0 51 77 154 109	252 168 0 0 0

^*Woods lattice lactate buffer solution also has 28mmol/L lactic acid, 4mmol/L K ⁺With 3mmol/L Ca ²⁺

When the inhalation, for example Refrigerant 12, trichlorofluoromethane, carbonic acid gas or other gas that is fit to are sent cupredoxin or cytopigment or its variant, derivative or structural equivalents from the packing of pressurization or atomizer with the form of aerosol spray can to use suitable propellent.Under the situation of pressurised aerosol, dose unit can be determined with the amount of sending metering by valve is provided.For example, be used for can be formulated as of sucker or insufflator and contain albumen and the suitable powder matrix such as the powdered mixture of lactose or starch as the capsule of gelatin and cartridge case.

When coming administration by topical, cupredoxin or cytopigment or its variant, derivative or structural equivalents can be formulated as solution, gelifying agent, ointment, ointment, suspensoid etc., as known in the art those.In some embodiments, by mode administration through the skin patch.When suppository (for example rectum or the vagina) administration, can also be in the composition that contains conventional suppository bases with cupredoxin and/or cytochrome c and its variant and derivative set of dispense.

When oral administration, can be by described cupredoxin or cytopigment or its variant, derivative or structural equivalents be easily prepared in cupredoxin or cytopigment or its variant, derivative or structural equivalents and pharmaceutically acceptable carrier well known in the art combination.Can adopt solid carrier, for example mannitol, lactose, Magnesium Stearate etc.; These carriers can make cupredoxin and/or cytopigment be configured to tablet, pill, lozenge, capsule, liquid agent, gelifying agent, syrup, slurries agent, suspensoid etc. with its variant and derivative, are used for the individual orally ingestible that will treat.For oral solid formulation, for example powder, capsule and tablet, suitable vehicle comprises weighting agent for example sugar, cellulosics, granulating agent and tackiness agent.

Well known in the art other easily carrier also comprise the multivalence carrier, for example bacterial capsule polysaccharide, dextran or genetically engineered carrier.In addition, the sustained release preparation that comprises cupredoxin or cytopigment or its variant, derivative or structural equivalents allows to discharge cupredoxin or cytopigment or its variant, derivative or structural equivalents in the long time, make when not having described sustained release preparation, then described cupredoxin or cytopigment or its variant, derivative or structural equivalents will cause or strengthen result of treatment before from the System Cleaning of individuality and/or by for example proteolytic enzyme and simple hydrolytic action degraded.

Can prolong or optimize the transformation period of composition of the present invention in blood flow by well known to a person skilled in the art certain methods, described method include but not limited to cyclisation peptide (Monk etal., BioDrugs 19 (4): 261-78, (2005); DeFreest et al., J.Pept.Res.63 (5): 409-19 (2004)), D, L-peptide (diastereomer), (Futaki et al., J.Biol.Chem.Feb 23; 276 (8): 5836-40 (2001); Papo et al., Cancer Res.64 (16): 5779-86 (2004); Miller et al., Biochem.Pharmacol.36 (1): 169-76, (1987)); Peptide (the Lee et al. that contains rare amino acid, J.Pept.Res.63 (2): 69-84 (2004)), end modified (the Labrie et al. of N-and C-, Clin.Invest.Med.13 (5): 275-8, (1990)), and hydrocarbon polymer nail pin (Schafmeister et al., J.Am.Chem.Soc.122:5891-5892 (2000); Walenski et al., Science305:1466-1470 (2004)).Interested especially is to replace or the d-isomerization (replacements) of L-aminoacid replacement and stabilized peptide sex modification and hydrocarbon polymer are followed closely pin through D-.

In various embodiments, described pharmaceutical composition comprises that carrier and vehicle (include but not limited to buffer reagent, carbohydrate, mannitol, protein, polypeptide or amino acid, for example glycine, antioxidant, fungistat, sequestrant, suspending agent, thickening material and/or sanitas), water, oil, salt brine solution, dextrose hydrate and glycerine solution, the required acceptable auxiliary substance of other pharmacy of approximate physiological condition is buffer reagent, tension regulator, wetting agent etc. for example.Recognize that though can adopt the known any suitable carrier administration composition of the present invention of those of ordinary skills, the type of carrier will depend on the mode of administration and change.Compound also can use technique known to be encapsulated in the liposome.Also can adopt biodegradable microballoon to be used for pharmaceutical composition of the present invention as carrier.For example, in United States Patent (USP) 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; The biodegradable microballoon that is fit to is disclosed in 5,814,344 and 5,942,252.

Described pharmaceutical composition can be sterilized by the known sterilising technology of routine, or can sterile filtration.The aqueous solution that obtains can packaged former state use, or by freeze-drying, freeze-dried preparation mixed with sterile solution before administration.

The administration of cupredoxin and/or cytopigment and its variant and derivative

Cupredoxin or cytopigment or its variant, derivative or structural equivalents can be formulated as pharmaceutical composition and come administration, and by any suitable approach for example by oral, suck, suction, hypogloeeis, rectum, vagina, per urethra, nose, part, through skin or parenteral (comprising in intravenously, intramuscular, the subcutaneous and coronary artery) administration.Its pharmaceutical preparation can be with any amount administration of effective its purpose of realization.More particularly, described composition is with the administration of treatment significant quantity.In specific embodiment, the treatment significant quantity generally is about 0.01-20mg/ day/kg body weight.

The compound that comprises cupredoxin or cytopigment or its variant, derivative or structural equivalents share individually or with other active groups in treating and/or preventing malaria infection.The character of the cupredoxin that the dosage that is fit to for example will depend on to be adopted certainly or compound, main body, the administering mode of cytopigment or its variant, derivative or structural equivalents and the illness that will treat and severity and change.Yet, usually, indicate the per daily dose of about 0.01-20mg/kg body weight in the people, to obtain gratifying result.The per daily dose of philtrum indication is that about 0.7mg is to about 1400mg cupredoxin or cytochrome c ₅₅₁Or the compound of its variant, derivative or structural equivalents, it conveniently gives with for example per daily dose, weekly dose, month dosage and/or successive administration.Per daily dose can be 1 to 12 time discontinuous dosage every day.Perhaps, dosage can be every other day, per three days, per four days, per five days, per six days, weekly and similarly fate increases to administration on the 31st.Perhaps, administration can continue, and it is undertaken by using patch, intravenous administration etc.

In some embodiments, method from structural equivalents to the patient that introduce cupredoxin or cytopigment or its variant, derivative or is undertaken by cupredoxin or cytopigment or its variant, derivative or structural equivalents and other Combined Preparation that is used for the medicine of malaria treatment.These methods are as known in the art.In specific embodiment, cupredoxin and/or cytochrome c be the cocktail (cocktail) that contains other malaria treatment agent or with the part of the Combined Preparation of other malaria treatment agent.Interested malaria treatment agent includes but not limited to chloroguanide, M-5943, Trimethoprim BP, chloroquine, Mefloquine hydrochloride, phenyl fluorenol, atovaquone, Pyrimethamine-Sulfadoxine, Pyrimethamine hcl-dapsone, halofantrine, quinine, Quinidine, Miaquin, amopyroquine, sulfanilamide (SN), Artemisinin, arteflene, Artemether, Artesunate, primaquine, Malaridine, chloroguanide, chloroquine, Mefloquine hydrochloride, Pyrimethamine-Sulfadoxine, Pyrimethamine hcl-dapsone, halofantrine, quinine, chloroguanide, chloroquine, Mefloquine hydrochloride, 1,16-ten hexa-methylenes (N-crassitude) dibromide and its combination.

In some embodiments, introduce cupredoxin or cytochrome c to the patient ₅₅₁Or the method for its variant, derivative or structural equivalents for example to contain the cocktail method of proteinase inhibitor identical with being used to introduce anti-HIV medicine at present.These methods are well known in the art.In specific embodiment, cupredoxin or cytochrome c ₅₅₁Its variant, derivative or structural equivalents be have anti-HIV therapeutical agent cocktail or with the part of anti-HIV therapeutical agent Combined Preparation.Anti-HIV medicine includes but not limited to reverse transcriptase inhibitors: AZT (zidovudine [Retrovir]), ddC (zalcitabine [Hivid], dideoxyinosine), d4T (stavudine [zerit]) and 3TC (lamivudine [Epivir]), non-nucleoside reverse transcriptase inhibitor (NNRTIS): Delavirdine (Rescriptor) and nevirapine (Viramune), proteinase inhibitor: ritonavir (Norvir), rltonavir and ritonavir combination (Kaletra), Saquinavir (Invirase), Indinavir vitriol (Crixivan), amprenavir (Agenerase) and viracept see nelfinaivr (Viracept).In some embodiments, the combination that will be called the several drugs of highly active antiretroviral therapy (HAART) is used for the treatment of described patient.

Determine correct preparation, route of administration and dosage by the attending doctor according to patient's illness.Dosage be can individually regulate and the active cupredoxin of result of treatment or the blood plasma level of cytopigment or its variant, derivative or structural equivalents are enough to keep to provide at interval.Usually, with the cupredoxin of expectation or cytopigment or its variant, derivative or structural equivalents with the mixture of pharmaceutical carrier in administration, described pharmaceutical carrier is put into practice according to the route of administration of desire meaning and standard drug and is selected.

On the one hand, described cupredoxin or cytopigment or its variant, derivative or structural equivalents are sent as DNA, made polypeptide produce in position.In one embodiment, as for example at Ulmer et al., described in (Science 259:1745-1749 (1993)) and Cohen (Science 259:1691-1692 (1993)) summarized, described DNA is " exposing ".Can be by DNA being coated on the picked-up that increases naked DNA on for example biodegradable pearl of carrier, described then carrier is by in the transporte to cells effectively.In these methods, DNA may reside among the known various delivery systems of those of ordinary skills any, and described system comprises expression of nucleic acid system, bacterium and virus expression systems.The technology that DNA is incorporated in these expression systems is known to a person of ordinary skill in the art.Referring to for example WO90/11092, WO93/24640, WO 93/17706 and United States Patent (USP) 5,736,524.

Can the carrier of genetic material from organism biography shuttle to organism be divided into two kinds of general types with being used for: cloning vector is plasmid replication or phage, and it has the necessary zone of propagation in the host cell that is fit to, and foreign DNA can insert wherein; Foreign DNA is replicated and breeds, and is the component of carrier as it.Use expression vector (for example plasmid, yeast or animal virus genome) that exogenous genetic material is introduced host cell or tissue to transcribe and to translate for example DNA of cupredoxin and/or cytopigment of foreign DNA.In expression vector, the DNA of importing by operationally with element for example promotor be connected, described element gives the host cell signal highly to transcribe the DNA of insertion.Some promotors are useful especially, inducible promoter for example, its to specificity factor reaction transcribes with controlling gene.Cupredoxin or cytopigment operationally are connected with inducible promoters and can react to control the expression of cupredoxin or cytopigment and its variant and derivative specificity factor with its variant and derivative polynucleotide.The example of typical inducible promoters comprise to alpha-interferon, heat stress, heavy metal ion and steroid for example glucocorticosteroid (Kaufman, Methods Enzymol.185:487-511 (1990)) and tsiklomitsin react those.The inducible promoters of other expectations comprise the cell that construct is wherein introduced be not endogenous those, in these cells, react when not excessive external source provides inductor.Usually, useful expression vector plasmid normally.Yet other forms of expression vector such as virus vector (for example replication defective retrovirus, adenovirus and adeno-associated virus) are also considered.

Expectation final result by used organism or cell and used carrier instructs carrier to select.Usually, carrier comprises signal sequence, replication orgin, marker gene, polylinker site, enhancer element, promotor and transcription termination sequence.For example, can with cupredoxin or cytopigment gene clone in the carrier that can in carrying plasmodial mosquito, transmit, in the mosquito body, duplicate to prevent parasite.The transferability of carrier will allow that cupredoxin/cytopigment propagate in the mosquito that has also infected plasmodial vicinity.

Determine correct preparation, route of administration and dosage by the attending doctor according to patient's illness.Can single adjusting dosage be enough to treat the patient and/or keep the active cupredoxin of result of treatment and/or the blood plasma level of cytopigment and its variant and derivative to provide with the interval.Usually, the cupredoxin of expectation and/or cytopigment and its variant and derivative can with the mixture of pharmaceutical carrier in administration, described pharmaceutical carrier is according to the route of administration and the standard drug choice of practice of desire meaning.Can use one or more physiology acceptable carriers that comprise vehicle and auxiliary agent to prepare pharmaceutical compositions for use of the present invention in the usual way, described carrier help cupredoxin and/or cytopigment and its variant and derivatives active agent processing, be used to suppress or stimulate the secretion of cupredoxin and/or cytopigment and its variant and derivative, perhaps their mixture is made spendable preparation on the therapeutics.

The test kit that comprises cupredoxin and/or cytochrome c and its variant and derivative

On the one hand, the invention provides test kit, it contains one or more the following materials in packing or container: (1) comprises the biological active composite of cupredoxin or cytopigment or its variant, derivative or structural equivalents; (2) pharmacy acceptable assistant or vehicle; (3) be used for the device of administration, for example syringe; (4) administration specification sheets.Also consider the two or more embodiments that are present in the same container in component (1)-(4) wherein.

On the other hand, the invention provides test kit, it contains one or more the following materials in packing or container: (1) comprises the biological active composite of cupredoxin or cytopigment or its variant, derivative or structural equivalents; (2) malaria treatment agent, it includes but not limited to chloroguanide, M-5943, Trimethoprim BP, chloroquine, Mefloquine hydrochloride, phenyl fluorenol, atovaquone, Pyrimethamine-Sulfadoxine, Pyrimethamine hcl-dapsone, halofantrine, quinine, Quinidine, Miaquin, amopyroquine, sulfanilamide (SN), Artemisinin, arteflene, Artemether, Artesunate, primaquine, Malaridine, chloroguanide, chloroquine, Mefloquine hydrochloride, Pyrimethamine-Sulfadoxine, Pyrimethamine hcl-dapsone, halofantrine, quinine, chloroguanide, chloroquine, Mefloquine hydrochloride, 1,16-ten hexa-methylenes (N-crassitude) dibromide; (3) pharmacy acceptable assistant or vehicle; (4) be used for the device of administration, for example syringe; (5) administration specification sheets.Also consider the two or more embodiments that are present in the same container in component (1)-(5) wherein.

In some embodiments, described test kit also is contained in the anti-HIV therapeutical agent in packing or the container.Interested anti-HIV therapeutical agent includes but not limited to reverse transcriptase inhibitors: AZT (zidovudine [Retrovir]), ddC (zalcitabine [Hivid], dideoxyinosine), d4T (stavudine [Zerit]) and 3TC (lamivudine [Epivir]), non-nucleoside reverse transcriptase inhibitor (NNRTIS): Delavirdine (Rescriptor) and nevirapine (Viramune), proteinase inhibitor: ritonavir (Norvir), rltonavir and ritonavir combination (Kaletra), Saquinavir (Invirase), Indinavir vitriol (Crixivan), amprenavir (Agenerase) and viracept see nelfinaivr (Viracept).In some embodiments, the combination that will be called the several drugs of highly active antiretroviral therapy (HAART) is used for the treatment of described patient.

When test kit is provided, the different components in the described composition can be packaged in the independent container, and mixes at once before use.The independent packing of these components can allow prolonged preservation and not have the loss function of active ingredient.

The reagent that comprises in the test kit can provide in the container of any kind, and described container makes the life-span of different components be held and do not adsorbed or change by the material of container.For example, the sealed glass ampoule can be included in neutrality, non-reactive gas for example cupredoxin and/or cytochrome c and its variant and derivative freeze-drying polypeptide or the polynucleotide or the buffer reagent of the following packing of nitrogen.Ampoule can for example glass, organic polymer such as polycarbonate, polystyrene etc., pottery, metal or common any other material that holds similar reagents that adopts constitute by any suitable material.Other examples of the container that is fit to comprise can be from the simple bottle of the similar material manufacturing of ampoule and can comprise paper tinsel for example aluminium or the inner involucrum of alloy lining.Other containers comprise test tube, bottle, flask, bottle, syringe etc.Container can have the aseptic hole that enters, and for example has the bottle of stopper, and described stopper can be by the subcutaneous injection needle-penetration.Other containers can have two chambers of being separated by the film of removing easily, and described film allows each several part to mix when removing.Removable film can be glass, plastics, rubber etc.

Test kit can also be furnished with illustrative material.Specification sheets can be printed on paper or other matrix, and/or can be used as electronically readable medium such as diskette, CD-ROM, DVD-ROM, Zip dish, videotape, mrt, flash memory device waits provides.Detailed specification sheets can physically not link with test kit; But, can guide the user to use the specified internet website of retail trader of producer or test kit or provide as e-mail.

The modification of cupredoxin and/or cytopigment

Cupredoxin or cytopigment can be changed to produce aforesaid variant and derivative from chemically modifying or carrying out genetics.These variants and derivative can be synthetic by standard technique.

Except the naturally occurring allelic variant of cupredoxin and cytopigment, can also in cupredoxin or cytopigment encoding sequence, introduce by sudden change and change, described sudden change causes the change of the aminoacid sequence of the cupredoxin of encoding or cytopigment, and this change changes the ability of the parasitemia in the red corpuscle that cupredoxin or cytopigment suppress malaria infection indistinctively." nonessential " amino-acid residue is can change in wild-type cupredoxin sequence and do not change the residue of biologic activity, and " essential " amino-acid residue is that this biologic activity is necessary.For example, conservative amino-acid residue is not especially accepted to change between the prediction cupredoxin, thereby is " essential ".

The amino acid that can not change the active conservative property replacement of polypeptide is well known in the art.Useful conservative property is substituted in table 3, shown in " the preferred replacement ".Wherein one type amino acid is fallen within the scope of the present invention by the replacement of the conservative property of another amino acid replacement of same type, as long as described replacement does not substantially change the biologic activity of described compound.

Table 3. preferably replaces

Original residue	Exemplary replacement	The preferred replacement
			Ala(A) Arg(R) Asn(N) Asp(D) Cys(C) Gln(Q) Glu(E) Gly(G) His(H) He(I) Leu(L) Lys(K) Met(M) Phe(F) Pro(p) Ser(S)	Val, Leu, He Lys, Gln, Asn Gln, His, Lys, Arg Glu Ser Asn Asp Pro, Ala Asn, Gln, Lys, Arg Leu, Val, Met, Ala, Phe, nor-leucine nor-leucine, Ile, Val, Met, Ala, Phe Arg, Gln, Asn Leu, Phe, Ile Leu, Val, Ile, Ala, Tyr Ala Thr	Val Lys Gln Glu Ser Asn Asp Ala Arg Leu Ile Arg Leu Leu Ala Thr

Thr(T) Trp(W) Tyr(Y) Val(V)

Ser Tyr, Phe Trp, Phe, Thr, Ser Ile, Leu, Met, Phe, Ala, nor-leucine

Ser Tyr Phe Leu

The non-conservation of the main body of structure example of influence (1) polypeptide backbone such as the side chain of β-lamella or alpha helical conformation, (2) electric charge, (3) hydrophobicity or (4) target site replaces the function that can modify cytotoxic factor.Character based on common side chain is divided residue in groups, and is as shown in table 4.Non-conservation replace to make the member with one of these classifications be exchanged into another kind of other.Can introduce conservative property replacement site or more particularly introduce the non-conservation site replacing.

Table 4. amino acid classification

Classification	Amino acid
		The destruction chain conformation aromaticity of the tart alkalescence of hydrophobic neutral hydrophilic	Nor-leucine, Met, Ala, Val, Leu, Ile Cys, Ser, Thr Asp, Glu Asn, Gln, His, Lys, Arg Gly, Pro Trp, Tyr, Phe

Can use methods known in the art for example oligonucleotide mediated (fixed point) mutagenesis, L-Ala scanning and PCR mutagenesis to prepare variant polypeptide.(Carter, Biochem be (1986) J.237:1-7 can to carry out site-directed mutagenesis on clone's DNA; Zoller and Smith, MethodsEnzymol.154:329-350 (1987)), box mutagenesis, restriction selects mutagenesis (Wells et al., Gene 34:315-323 (1985)) or other known technology, to produce cupredoxin or cytochrome c ₅₅₁Modification D NA.

Also can use cupredoxin and cytochrome c ₅₅₁Known mutations produce in the method for the invention variant cupredoxin and the cytochrome c that uses ₅₅₁For example, the C112D of known azurin and M44KM64E mutant have cytotoxic activity and the cessation of growth cessation activity that is different from natural azurin, and the activity of this change can be used for methods of treatment of the present invention.An embodiment utilization of method of the present invention keeps cupredoxin and/or cytopigment and its variant and the derivative that suppresses the ability of the growth of malaria infection in the mammalian cell.In another embodiment, the cupredoxin variant for example M44KM64E mutant of method utilization of the present invention with the ability that causes the cell cessation of growth cessation.

More complete understanding of the present invention can be by obtaining with reference to following specific embodiment.For illustrative purpose rather than limit the scope of the invention, embodiment has been described individually.Because environment can be pointed out or it is suitable to make, so the present invention considers the replacement of variation and Equivalent.Though this paper has adopted specific term, these terms are intended that descriptive meaning, rather than the purpose in order to limit.Do not deviating under the spirit and scope of the present invention prerequisite, can carry out aforesaid modification of the present invention and variant, thereby only these restrictions in addition shown in appended claim.

Embodiment

Embodiment 1: cupredoxin and cytopigment are to the vitro inhibition of Plasmodium falciparum parasites mass formed by blood stasis

Cupredoxin bacterium wt azurin, M44KM64E azurin, iron sulphur bacterium azurin and cyanobacteria plastocyanin and cytopigment Pseudomonas aeruginosa cytochrome c ₅₅₁, human cytochrome c and bedding seat frustule pigment f under the 200 μ g/ml concentration in test in normal red corpuscle (RBC) is measured in 30 hours behind the incubation.In these experiments, with normal RBC serum free medium washed twice, and resuspended with complete RPMI to 10% hematocrit.200 μ l 10%Hct RBC are added into (final 2%Hct 1mL), adds the complete RPMI of 30 μ l in addition, and it contains 666 μ M final concentrations is reorganization cupredoxin or the cytochrome protein of 200 μ M in each hole in 24 holes.Culture prepared the parasite in schizont stage in 10 minutes centrifugal late period under 3200rpm by the Percoll cushion.For infecting, in the time of t=0 hour, add 4 * 10 of 500 μ l volumes ⁶Individual parasite/hole.With plate incubation 30 hours, score by thin blood smear and Giemsa staining at this moment.

Contrast shows 9.5% parasitemia (standard error 1.3%), wt azurin 6.9% (s.e.1.4%), M44KM64E azurin 9.1% (s.e.1.0%), iron sulphur bacterium azurin 7.2% (s.e.0.7%), cytochrome c ₅₅₁7.5% (s.e.1.5%), human cytochrome c 8.4% (s.e.0.4%), plastocyanin 8.1% (s.e.1.3%) and cytopigment f 6.6% (s.e.1.0%), this shows cupredoxin for example wt azurin and iron sulphur bacterium azurin, and cytopigment cytochrome c for example ₅₅₁Show and suppress parasitemia 20 to 30%.

When each stage of parasite life cycle (0-24 hour, ring bodies (ringformation); 24-36 hour, trophont; 36-48 hour, schizont) when testing cupredoxin, contrast shows that average 0.1% ring bodies and 9.4% trophont form, and the wt azurin shows there is not ring bodies, and 6.9% trophont forms; Cytopigment f shows 0.2% ring bodies, forms and have significantly low (6.3%) trophont.Significantly, iron sulphur bacterium azurin shows very high (2.0%) ring bodies, and significantly reduced (5.2%) trophont forms.Other not significantly effect.Parasite in the sample sets that iron sulphur bacterium azurin is handled seems ill with comparing of all the other sample sets and is dying that this shows that iron sulphur bacterium azurin has remarkable inhibition and toxic action to the parasite growth.

Embodiment 2: iron sulphur bacterium azurin is to the vitro inhibition of plasmodium falciparum time multiplexed cell system

For determining whether bacterial oxidation reduction albumen can suppress plasmodial time multiplexed cell system, use hypotonic blood shadow goods red corpuscle to be loaded into recombinant protein concentration in the cell of 200 μ g/ml.As described in example 1 above, washed cell, parasite (plasmodium falciparum) resuspended, the also usefulness schizont stage infect then.With erythrocyte ghost incubation 19 hours and 40 hours, make Jim Sa smear.

Compare with normocytic infection among the embodiment 1, in loaded cells blood shadow culture, only iron sulphur bacterium azurin reduces total parasitemia.In the time of 19 hours, in intrusion and ring formation, there is not significant difference, empty blood shadow is 5.0 ± 0.4%, it is 4.5 ± 1.0% that iron sulphur bacterium azurin loads blood shadow.Yet in the time of 40 hours, iron sulphur bacterium azurin loads blood shadow and has lower level infection.Use any bacterioprotein in the time of 19 hours, not see main effect.Yet, in the time of 40 hours, contrasts the blood shadow that is untreated and show 4.6 ± 0.3% parasitemia, and iron sulphur bacterium azurin processing blood shadow has 2.7 ± 0.8% parasitemia, almost reduces by 50%.Referring to table 5.Wt azurin, M44KM64E mutant azurin, plastocyanin, cytochrome c ₅₅₁, human cytochrome c and cyanobacteria cytopigment f albumen shows from 4.2 to 5.4% parasitemia.

Table 5. cupredoxin and cytopigment are to the inhibition of the falciparum infection of erythrocyte ghost

Handle	Mean parasitized worm mass formed by blood stasis in the time of 40 hours	Standard error
			Empty wild-type azurin M44KM64E azurin iron sulphur bacterium azurin cytochrome C 551 human cytochrome c plastocyanin cytopigment f	4.6％ 5.4％ 4.7％ 2.7％ 4.2％ 4.6％ 4.3％ 4.5％	0.3％ 1.0％ 0.5％ 0.8％ 0.4％ 0.8％ 0.3％ 0.9％

Embodiment 3: azurin and and the Fab fragment of PfMSP1-19 compound G17.12 monoclonal antibody between structural homology

Research in the past shows, the structural similarity of cupredoxin demonstration and immunoglobulin superfamily member's variable region.(Gough & Chothia，Structure12：917-925(2004)；Stevens et al.，J.Mol.Recognit.18：150-157(2005))。Use DALI algorithm (Holm ﹠amp; Park, Bioinformatics 16:566-567 (2000)).Search is from the structural similarity of the azurin (1JZG) of Pseudomonas aeruginosa in the 3D database.Azurin show and with the segmental structural similarity of Fab of the Fab fragment compound G17.12 monoclonal antibody of the PfMSP1-19 fragment G17.12 monoclonal antibody of the MSP1 schizont surface protein of plasmodium falciparum.(Pizarro et al.，J.Mol.Biol.328：1091-1103(2003))。(table 6) azurin also shows the structural similarity (table 6) with ICAM-1, and ICAM-1 participates in cerebral malaria and may relate to acceptor on the erythrocytic endotheliocyte that is used to detain falciparum infection in the capillary blood vessel culture as brain and its hetero-organization.(Smith et al.，Proc.Natl.Acad.Sci.USA 97：1766-1771(2000)；Franke-Fayard et al.，Proc.Natl.Acad.Sci.USA102：11468-11473(2005))。

This embodiment shows that the cupredoxin that comprises azurin shows structural similarity: have two face-to-face assembling and the antiparallel β lamellas that connected by disulfide linkage, it is connected with immunoglobulin superfamily member's variable region and the cell foreign lands of intercellular adhesion molecule (ICAM) and their part.

The structural similarity of table 6. Pseudomonas aeruginosa azurin and Different types of etiopathogenises associated protein

PDB	Note	Reference	Reddish black egg (1jzg)
				The DALIz score value (1)
			1VCAB1 1ZXQ1 1IAM1 1OB1A1 1topB 2NCM		People's vascular cell adhesion molecule-1, the structure of two N-terminal structural domains of the crystalline structure ICAM-1 of VCAM-1 ICAM-2 and the crystalline structure ICAM-3 of plasmodium falciparum MSP1-19 compound Fab mixture and the composite structure nerve cell adhesion molecule that combines the territory of Alpha β 2, NCAM	17 19 20 21 22 23	3.5 3.3 3.0 2.9 2.5 2.4

⁽¹⁾-use DALI (16) carries out the structure parallelism with azurin.The structure of DALIz score value＜2 is to being considered to dissimilar.

Embodiment 4:Laz and the H.8-clone and the expression of azurin fusion gene

The laz gene of Diplococcus gonorrhoeae is cloned according to its known array (SEQ ID NO:22).Use be called the Pseudomonas aeruginosa azurin gene (SEQ ID NO:1) of paz and Diplococcus gonorrhoeae (N.gonnerrhoeae) laz H.8 epi-position sequence (SEQ ID NO:21) by reading frame H.8 epitope gene be cloned into 5 of paz '-end to produce H.8-paz or to be cloned into 3 of paz '-end with generation paz-H.8.

Table 7. cancer cells, bacterial isolates and gene construct

Cell/bacterial strain/plasmid	Relevant feature *	Reference
			Pseudomonas aeruginosa PAO1 E.coli JM109 E.coli BL21 (DE3) NEISSERIA GONORRHOEAE F62 pUC18 pUC19 pUC18-laz pUC19-paz pUC18-H.8-paz pGEX-5X-3 pET29a pET29a-gst pGEX-5X-3-H.8 pET29a-gst-H.8	Prototroph, FP-(sex factor defective) clone and azurin are expressed strain GST expression strain and are used for the general cloning vector of the isolating prototroph of DNA, Ap rGeneral cloning vector, Ap rBe cloned among the pUC18 from the 1kb PCR fragment cloning of the genomic dna of Diplococcus gonorrhoeae F62 the 0.55kb fragment in the pUC19 of HindIII and PstI digestion, Ap from Pseudomonas aeruginosa PAO1 rCoding from Diplococcus gonorrhoeae H.8 with from the fusion plasmid of the azurin of Pseudomonas aeruginosa PAO1, Ap rThe gst gene fusion vector, Ap rThe E.coli expression vector, Km rThe pET29a derivative that contains the gst gene, Km rContain the H.8 pGEX-5X-3 derivative of coding region, Ap rThe pET29a derivative that contains the gst-H.8 gene, Km r	Hollowy, et al., Microbiol.Rev. 43:73-102 (1979) Yanisch-Perron, et al., Gene 33:103-119 (1985) Nvagen American Type Culture Collection Yanisch-Perron, et al., the same Yanisch-Perron, et al., the same Yamada herein, et al., Proc.Natl.Acad.Sci. USA 99:14098-14103 (2002); Amersham Novagen is herein herein for Yamada, et al .Proc.Natl.Acad.Sci. USA 101:4770-4775 (2004)

^*Ap, penbritin; Km, kantlex; GST, glutathione-S-transferase.

The clone of paz and laz gene and expression

The clone and the overexpression of azurin gene have been described.(Yamada，et al.，Proc.Natl.Acad.Sci.USA 99：14098-14103(2002)；Punj，et al.，Oncogene 23：2367-2378(2004))。The genomic dna that uses Diplococcus gonorrhoeae strain F62 by PCR is as the increase Laz encoding gene (laz) of Diplococcus gonorrhoeae of template DNA.The forward and the reverse primer that use are

5 '-CCG GAATTCCGGCAGGGATGTTGTAAATATCCG-3 ' (SEQ ID NO:23) and

5 '-GG GGTACCGCCGTGGCAGGCATACAGCATTTCAATCGG-3 ' (SEQ ID NO:24), wherein EcoRI and the KpnI restriction site of introducing in addition marks with underscore respectively.To be inserted into pUC18 carrier (Yanisch-Perron with the amplification of DNA fragments of the 1.0kb of EcoRI and KpnI digestion, et al., Gene 33:103-119 (1985)) in the corresponding site, make the laz gene be positioned at the downstream of lac promotor, to produce expression plasmid pUC18-laz (table 7).

With pUC19-paz and pUC18-laz as template by PCR construction expression Diplococcus gonorrhoeae Laz H.8 with the plasmid of azurin (Paz) syzygy of Pseudomonas aeruginosa.For syzygy H.8-Paz, use pUC18-laz as template and following primer amplification 3.1kb fragment: 5 '-(phosphorylation) GGCAGCAGGGGCTTCGGCAGCATCTGC-3 ' (SEQID NO:25) and 5 '-CTGCAG GTCGACTCTAGAGGATCCCG-3 ' (SEQ IDNO:26), wherein the salI site marks with underscore.The 0.4kb fragment of pcr amplification obtains from pUC19-paz and the following primer as template: 5 '-(phosphorylation) GCCGAGTGCTCGGTGGACATCCAGG-3 ' (SEQ ID NO:27) and 5 '-TA CTCGAGTCACTTCAGGGTCAGGGTG-3 ' (SEQ ID NO:28), wherein the XhoI site marks with underscore.Clone's SalI digestion from the PCR fragment of pUC 18-laz and XhoI digestion from the PCR fragment of pUC19-paz to produce expression plasmid pUC18-H.8-paz (table 7).

E.coli JM109 is used the host strain that acts on expression azurin and its derivative gene.Reorganization E.coli bacterial strain is containing 100 μ g/ml penbritins, 0.1mM IPTG and 0.5mM CUSO ₄2X YT substratum in cultivate down 16h to produce azurin protein at 37 ℃.

When the E.coli bacterial strain that carries these plasmids is cultivated in the presence of IPTG, lysing cell and as (Yamada, et al., Proc.Nat1.Acad.Sci.USA99:14098-14103 (2002) as described in to azurin; Punj, et al., Oncogene 23:2367-2378 (2004); Yamada, et al., Cell.Microbiol.7:1418-1431 (2005)) purifying protein, various azurin derivatives move as single component on SDS-PAGE, (Cannon, Clin.Microbiol.Rev.2:S 1-S4 (1989) but as previously noted; Fisette, et al., J.Biol.Chem.278:46252-46260 (2003)), contain the migration that shows abnormality of H.8 albumen (about 17kDa).

Merge the proteic plasmid construction of GST

The plasmid of wt azurin (azu) derivative of polymerase chain reaction construction expression syzygy glutathione S-transferase (the GST)-brachymemma by use proofreading and correct archaeal dna polymerase.For pGST-azu 36-128, the amplification PCR fragment is introduced commercial GST expression vector pGEX-5X, and (Amersham Biosciences, Piscataway is in BamH1 NJ) and the EcoR1 site.Use pUC19-azu as template and following primer amplification fragment: 5 '-C GGGATCCCCG GCA ACC TGC CGA AGA ACG TCA TGG GC-3 ' (SEQ ID NO:29) and 5 '-CG GAATTCGCA TCA CTT CAG GGT GAGGG-3 ' (SEQ ID NO:30), wherein BamH1 and the EcoR1 restriction site of introducing in addition marks with underscore respectively.(Stratagene, La Jolla CA) introduce terminator codon and carry out cumulatively the C-terminal clipped form of azu gene by using QuickChange site-directed mutagenesis test kit.

For pGST-azu 36-89, terminator codon is introduced among the Gly90.Use is carried the plasmid of pGST-azu 36-128 as template DNA.Three groups of oligonucleotide that are used for site-directed mutagenesis show below.For pGST-azu 36-89:5 '-CCA AGC TGA TCG GCT CGTGAG AGAAGG ACT CGG TGA CC-3 ' (SEQ ID NO:31) and 5 '-GGTCAC CGA GTC CTT CTC TCA CGA GCC GAT CAG CTT GG-3 ' (SEQID NO:32).

For pGST-azu 88-113, (Stratagene, La Jolla CA) introduce terminator codon and carry out cumulatively the C-terminal clipped form of azu gene by using QuickChange site-directed mutagenesis test kit.For pGST-azu 88-113, terminator codon is introduced among the Phel 14.Use is carried the plasmid of pGST-azu 88-128 as template DNA.For pGST-azu88-128, the amplification PCR fragment is introduced among the BamH1 and EcoR1 site of commercial GST expression vector pGEX-5X (Amersham Biosciences).Use pUC 19-azu as template and following primer amplification fragment: 5 '-C GGGGATCCCCG GCT CGGGCG AGA AGG AC-3 ' (SEQ ID NO:33) and 5 '-CGG GAATTCTCC ACTTCA GGG TCA GGG TG-3 ' (SEQ ID NO:34), wherein BamH1 and the EcoR1 restriction site of introducing in addition marks with underscore respectively.

For the preparation of pGST-azu 88-113, one group of oligonucleotide that is used for site-directed mutagenesis shows below: 5 '-GTT CTT CTG CAC CTA GCC GGG CCA CTC CG-3 ' (SEQID NO:35) and 5 '-CGG AGT GGC CCG GCT AGG TGC AGA AGAAC-3 ' (SEQ ID NO:36).Use pGST-azu 88-113 Transformed E .coli XL-1Blue bacterial strain.Use commercial reagents box (Qiagen, Venlo, The Netherlands) to carry out plasmid and extract, carry out PCR and check order and estimate plasmid and insert and transfection.

Use E.coli BL21 (DE3) as the host strain that is used to express gst and its syzygy derivative.The E.coli strain X Ll-Blue that transforms with the pGST-azu plasmid cultivated three hours down at 37 ℃ in having the LB substratum of penbritin, carry out IPTG (0.4mM) this moment and induce, subsequently at 37 ℃ of following incubation 2-4h so that the expression level maximization.By centrifugal separating cell, and be resuspended in 25mL 1 * PBS damping fluid.Lysis subsequently comprises two processing continuously of heat-cold shock (using suitable proteinase inhibitor) in supersound process cell suspension (20 minutes) and acetone-the dry ice bath on ice.The supernatant liquor of isolated cell cleavage mixture, and with it by fresh filling and PBS equilibrated 1mL gsh-sepharose 4B (AmershamBiosciences) post.Post washing and being used in after the 10mM gsh wash-out GST-azu product among the 20mM Tris-HCl pH 8. subsequently, use 10%SDS-PAGE Tris-Gly gel with Xylene Brilliant Cyanine G R reagent dyeing by electrophoresis test GST-Azu 88-113 purity.Use the Bradford method to measure protein concentration.

Embodiment 5: azurin combines with C-terminal fragment MSP1-19 and the MSP1-42 of plasmodium falciparum schizont surface protein MSP1.

Consider azurin and and the Fab fragment of Pf MSP1-19 compound monoclonal antibody G17.12 between structural similarity (table 6) (Pizarro et al., the same), measure the ability of azurin and PfMSP1-42 or PfMSP1-19 formation mixture.Two kinds of derivative L az of test days green white and wherein the H.8 epi-position of Laz be fused to the H.8-azurin of the N-terminal portions (as described in example 4 above) of Pseudomonas aeruginosa azurin by reading frame, described Laz be from neisserial (gonnococci) and meningococcus (meningococci) as having of Neisseria meningitidis other be called H.8 39 amino acid epi-position (Gotschlich ﹠amp of epi-position; Seiff, FEMS Microbiol.Lett.43:253-255 (1987); Kawula et al., the azurin sample albumen of Mol.Microbiol.1:179-185 (1987).

Use is estimated external protein-protein interaction from the Biacore X-ray spectrometer of Biacore AB International.All experiments use Au-CM5 sensor chip (Biacore) to carry out under 25 ℃ in HBS-EP electrophoretic buffer (0.01M HEPES, pH 7.4,0.15M NaCl, 3mMEDTA, 0.005%v/v tensio-active agent P20).Carry out the immobilization of albumen on the CM5 chip according to the amine coupling step.Immobilization albumen after the pre-activation of the NHS/EDC on CM5 surface: inject 50 μ l azurins (510 μ M).Use thanomin (1M, pH 8.8) to handle the CM5 surface subsequently to remove uncrosslinked albumen.Inject albumen elutriant (50 μ l) with 30 μ l/ minutes flow velocitys on albumen-CM5 surface, 120 seconds time lag when injection finishes is carried out combination research.The albumen elutriant comprise GST-azurin fusion rotein (GST, GST-Azu 36-128, GST-Azu 36-89 and GST-Azu 88-113, as described in example 4 above).Between protein injection, use 100mM NaOH (10 μ l injection pulse) reg sensor chip surface.All comes parallel carrying out in conjunction with research with respect to the negative fluid channel of using naked Au-CM5 sensor surface, with the non-specific combination of correction with chip.For producing the binding constant data, the albumen elutriant (0.05-2000nM) that increases concentration by injection designs titration experiments.As illustrated in the Biacore software, with the SPR data fitting to Langmuir (1:1) balance combination model [Req=Rmax/ (1+Kd/C], the binding constant (Kd) of therefrom extrapolating.

By surface plasma resonance (SPR) assay determination Pf MSP1-19 and Pf MSP1-42 albumen and azurin, H.8-the specificity of azurin and Laz interacts, data are shown in Figure 1.The bonded SPR influence chart of being used for fixing PfMSP1-19 and PfMSP1-42 and azurin and its derivative shows the selectivity identification among these albumen.Though the nanomolar concentration of azurin allows significantly to combine with immobilization MSP1-19 (accompanying drawing 1A) or MSP1-42 (accompanying drawing 1B), but H.8-azurin and Laz show the higher binding affinity with schizont surface protein MSP1 split product, characteristic Kd value between azurin and the MSP1-19 is 32.2nM, is 54.3nM between azurin and the MSP1-42.H.8-the Kd value between azurin and MSP1-19 and the MSP1-42 is 11.8nM and 14.3nM, and these values between Laz and MSP1-19 and MSP1-42 are respectively 26.2nM and 45.6nM.

For checking whether epi-position H.8 can promote combining of azurin H.8-or Laz and PfMSP1-19 or PfMSP1-42 part, tested glutathione S-transferase (GST) with wherein H.8 epi-position be fused to GST N-end (referring to embodiment 4) the fusion derivative H.8-GST with MSP1-19 or MSP1-42 bonded ability.GST or H.8-GST debond PfMSP1-19 (accompanying drawing 1A) or MSP1-42 (accompanying drawing 1B) combine but H.8-GST demonstrate with the weak of MSP1-42.

Glutathione s-transferase (GST) and some fusion roteins (Yamada et al., Cell.Microbiol.7:1418-1431 (2005) and embodiment 4) and the MSP1-19 bonded ability that merge of part and the GST of azurin have wherein been tested.Independent GST or wherein the azurin aminoacid sequence 88 to 113 in 128 amino acid of azurin do not show any combination (accompanying drawing 1C) by reading frame with the GST-Azu 88-113 that GST merges, and the GST-Azu36-128 that has the GST-Azu 36-89 of aminoacid sequence 36 to 89 and have amino acid 36 to 128 demonstrates with the remarkable of MSP1-19 and combines, and the Kd value is respectively 20.9nM and 24.5nM.

Embodiment 6: azurin, H.8-azurin and Laz be to the inhibition of plasmodium falciparum

Use schizont stage parasite and normal red corpuscle (RBC) are measured the degree of parasitemia.With normal red corpuscle (RBC) washed twice in serum free medium, and resuspended with complete RPMI to 10% hematocrit.The RBC of 200 μ l, 10% hematocrit is added in each hole in 24 holes, adds in addition and contain or do not contain the azurin of different concns, the complete RMPI of 300 μ l of azurin or Laz H.8-.Culture prepared the Plasmodium falciparum parasites in schizont stage in 10 minutes centrifugal late period under 3200rpm by the Percoll cushion.For infecting, add 4 * 10 of 500 μ l volumes at the zero-time point ⁶Individual parasite/hole.Plate is incubated overnight (about 16h), scores by thin blood smear and Giemsa staining at this moment.

Although under high relatively concentration (about 50 μ M), azurin, H.8-azurin or the Laz significant parasitemia that all shows the dose-dependently mode suppresses (accompanying drawing 2).Infer that this high density has reflected that plasmodium invades erythrocytic number of ways (Cowman et al., FEBS Lett.476:84-88 (2000); Baum et al., J.Biol.Chem.281:5197-5208 (2006)), the azurin of high density or Laz disturb invasion procedure necessary.To as indicated in the enhanced binding affinity of MSP1-19, H.8-azurin and Neisser Laz albumen are compared the inhibition (accompanying drawing 2) of the Plasmodium falciparum parasites mass formed by blood stasis that demonstrates higher level with azurin as them.

When using with red fluorescence dyestuff Alexa fluor 568 mark azurins and in intrusion is measured, in RBC, almost detect less than red fluorescence, as if this shows that azurin does not enter the part of RBC as bonded MSP1-19, more possibly, show RBC that schizont exists be wherein azurin can not with the MSP1-19 bonded those.These data meet observation (the Yamada et al. before us fully, Cell.Microbiol.7:1418-1431 (2005)), be that azurin does not enter normal cell for example scavenger cell, mastocyte etc., azurin, H.8-the effect of azurin or Laz is on the intrusion level rather than on the parasitic time multiplexed cell system.In a word, data sheet green white tomorrow among Fig. 2, H.8-the potential anti-malarial effect of azurin and Laz is by disturbing the intrusion of parasite to RBC.

Embodiment 7: azurin is in conjunction with ICAM

Interesting structural similarity (table 6) (Wassmer et al., PloS Med.2:885-890 (2005) between azurin and the known ICAM that participates in as the erythrocytic acceptor of falciparum infection; Dormeyer et al., Antimicrob.Agents Chemotherap.50:724-730 (2006)) impel and carry out between azurin and the ICAM as the test analysis of the measured protein-protein interaction of SPR, described ICAM is ICAM-1, ICAM-2, ICAM-3 and NCAM for example.Use is immobilized azurin on the CM5 chip, ICAM-3 (accompanying drawing 3, Kd=19.5+5.4nM) and NCAM (accompanying drawing 3, illustration) shown strong combination, but what is interesting is that ICAM-1 and ICAM-2 do not show.Though be not limited to the mode that the present invention is worked, azurin also may be that interaction by itself and ICAM-3 or NCAM mediates for the partial action that suppresses the Plasmodium falciparum parasites mass formed by blood stasis.

Embodiment 8: may be exposed to or be exposed to the patient's of malaria treatment

The medicine that comprises one or more cupredoxins and/or cytopigment that will be used for the clinical use of prevention of malaria gives the patient.

Not to blood stage Plasmodium falciparum parasites to deposit antibody history (measuring as immunofluorescence assay) earlier but live in malaria be the pharmaceutical preparation that 15 healthy male volunteers in the age 22-50 year in the endemic area will be accepted the cytopigment of the cupredoxin of injection purifying and purifying.Two such people will be as untreated contrast.

Aseptic pharmaceutical preparation is the form that is designed for the single dosage ampoule of 0.5ml of the aseptic Pseudomonas aeruginosa azurin in the pharmaceutical preparation of intravenous administration, and this is well known to a person skilled in the art.Pharmaceutical preparation is kept under 4 ℃, before administration, keeps in Dark Place.In a clinical trial, azurin is prepared into five different concns: 10 μ g, 30 μ g, 100 μ g, 300 μ g and 800 μ g azurin/0.5ml dosage.

Through intravenously to 13 these pharmaceutical preparations of volunteer's administration, every 10 dosage.The volunteer accepted first treatment on 0th, three weeks were accepted identical dosage every other day subsequently.The injection back was observed volunteer's instant toxic action in 20 minutes.After 24 and 48 hours, check the evidence of their fever, local tenderness, erythema, heating, sclerosis and lymph node pathological change, inquiry is about the main suit of headache, heating, shiver with cold, discomfort, local pain, nauseating and arthralgia.Before each administration, gather blood and urine sample and be used for complete laboratory examination.After each administration, reexamined full blood count and serum chemistry feature in two days.(Portland ME) detects plasmodial existence for Binax, Inc. for optical microphotograph microscopy (ME) by painted blood smear or ICT malaria P.f/P.v. test kit.The result shows the validity of treatment.

Embodiment 9: the control of the malaria infection of insect

To be operably connected to the cupredoxin encoding sequence that is operably connected with constitutive promoter from the genetic elements propagated that a mosquito is passed to the another mosquito.Thereby the Pseudomonas aeruginosa azurin will produce in by the malarial mosquito of falciparum infection, and will disturb plasmodium falciparum duplicating in mosquito/survive.Then this mosquito is introduced the endemy area, make the genetic elements of carrying azurin will propagate in the malarial mosquito of other falciparum infections, suppress plasmodium falciparum growth or survival.

Embodiment 10: the patient's of malaria infection treatment

The clinical use medicine of malaria treatment that will comprise one or more cupredoxins and/or cytopigment is used to treat the malaria infection of philtrum.

The pharmaceutical preparation of Pseudomonas aeruginosa azurin of 15 healthy male volunteers usefulness purifying with age 22-50 year of depositing antibody history (analyzing institute's mensurations as immunofluorescence technique) earlier of blood stage Plasmodium falciparum parasites is injected.Two such people are as untreated contrast.

Aseptic pharmaceutical preparation is the form that is designed for the single dosage ampoule of 0.5ml of the aseptic Pseudomonas aeruginosa azurin in the pharmaceutical preparation of intravenous administration, and this is well known to a person skilled in the art.Pharmaceutical preparation is kept under 4 ℃, keeps in Dark Place before the administration.In a clinical trial, the Pseudomonas aeruginosa azurin is prepared into five different concentration: 10 μ g, 30 μ g, 100 μ g, 300 μ g and 800 μ g azurin/cytochrome cs ₅₅₁(in molecular basis 1: 1)/0.5ml dosage.

Through intravenously to 13 these pharmaceutical preparations of volunteer's administration, 10 doses every.The volunteer accepted first treatment on 0th, three weeks were accepted identical dosage every other day subsequently.The injection back was observed volunteer's instant toxic action in 20 minutes.After 24 and 48 hours, check the evidence of their fever, local tenderness, erythema, heating, sclerosis and lymph node pathological change, inquiry is about the main suit of headache, heating, shiver with cold, discomfort, local pain, nauseating and arthralgia.Before each administration, gather blood and urine sample and be used for complete laboratory examination.After each administration, reexamined full blood count and serum chemistry feature on the 2nd.(Portland ME) measures plasmodial existence for Binax, Inc. for optical microphotograph microscopy (ME) by painted blood smear or ICT malaria P.f./P.v. test kit.The result shows the validity of treatment.

Claims (41)

1. isolating peptide, it is variant, derivative or the structural equivalents of cupredoxin or cytopigment, and it can suppress the parasitemia of malaria in the red corpuscle of malaria infection.

2. the isolating peptide of claim 1, it can suppress the parasitemia of malaria in the HRBC that plasmodium falciparum (P.falciparum) infects.

3. isolating peptide, it is variant, derivative or the structural equivalents of cupredoxin or cytopigment, and it can suppress plasmodial time multiplexed cell system in the HRBC of malaria infection.

4. isolating peptide, it is variant, derivative or the structural equivalents of cupredoxin, and it can be in conjunction with the albumen that is selected from PfMSP1-19 and PfMSP1-42.

5. the isolating peptide of claim 1, wherein said cupredoxin are selected from azurin, false azurin, plastocyanin, iron sulphur bacterium azurin, Laz and the green element that deflects of orange.

6. the isolating peptide of claim 5, wherein said cupredoxin chosen from Fe sulphur bacterium azurin, azurin and Laz.

7. the isolating peptide of claim 1, wherein said cupredoxin is from organism, and described organism is selected from Pseudomonas aeruginosa Pseudomonas aeruginosa), Alcaligenes faecalis (Alcaligenes faecalis), Achromobacter xylosoxidans (Achromobacter xylosoxidan), bordetella bronchiseptica (Bordetella bronchiseptica), methylomonas (Methylomonas sp.), Neisseria meningitidis (Neisseria meningitidis), Diplococcus gonorrhoeae (Neisseria gonorrhea), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas chlororaphis (Pseudomonas chlororaphis), xyllela fastidiosa (Xylella fastidiosa) and Vibrio parahaemolyticus (Vibrio parahaemolyticus).

8. the isolating peptide of claim 6, it is from organism, and described organism is selected from thiobacillus ferrooxidant (Thiobacillus ferrooxidans), Pseudomonas aeruginosa, Diplococcus gonorrhoeae and Neisseria meningitidis.

9. the isolating peptide of claim 1, wherein said cytopigment are selected from cytochrome c and cytopigment f.

10. the isolating peptide of claim 9, wherein said cytochrome c is from organism, and described organism is selected from people and Pseudomonas aeruginosa.

11. the isolating peptide of claim 9, wherein said cytopigment f is from cyanobacteria (cyanobacteria).

12. the isolating peptide of claim 1, wherein the clipped form of peptide is selected from SEQ IDNO:1-20 and 22.

13. the isolating peptide of claim 1, it has at least 90% aminoacid sequence homogeny with the sequence that is selected from SEQ ID NO:1-20 and 22.

14. the isolating peptide of claim 1, it is the clipped form of cupredoxin or cytopigment.

15. the isolating peptide of claim 1, wherein said peptide surpasses about 10 residues, and is no more than about 100 residues.

16. the isolating peptide of claim 1, wherein said peptide comprise azurin residue 36-89.

17. the isolating peptide of claim 1, wherein said peptide is made of azurin residue 36-89.

18. the isolating peptide of claim 1, wherein said peptide comprise cupredoxin and residue azurin 36-89 equivalence.

19. the isolating peptide of claim 1, it is fused to the H.8 district of Laz.

20. the isolating peptide of claim 1, it is the structural equivalents of monoclonal antibody G17.12.

21. a composition, it is contained in the isolating peptide of at least a cupredoxin, cytopigment or claim 1 in the pharmaceutical composition.

22. the composition of claim 21 is wherein prepared described pharmaceutical composition for intravenous administration.

23. the composition of claim 21, it comprises another kind of antimalarial drug in addition.

24. the composition of claim 21, it comprises anti-HIV medicine in addition.

25. the composition of claim 21, wherein said cupredoxin is from organism, and described organism is selected from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidans, bordetella bronchiseptica, methylomonas, Neisseria meningitidis, Diplococcus gonorrhoeae, Pseudomonas fluorescens, Pseudomonas chlororaphis, xyllela fastidiosa and Vibrio parahaemolyticus.

26. the composition of claim 25, wherein said cupredoxin are from organism, described organism is selected from thiobacillus ferrooxidant, Pseudomonas aeruginosa, Diplococcus gonorrhoeae and Neisseria meningitidis.

27. the composition of claim 21, wherein said cytopigment are selected from cytochrome c and cytopigment f.

28. the composition of claim 27, wherein said cytochrome c are from organism, described organism is selected from people and Pseudomonas aeruginosa.

29. the composition of claim 27, wherein said cytopigment f is from cyanobacteria.

30. the composition of claim 21, wherein said cupredoxin or cytopigment are selected from SEQ ID NO:1-20 and 22.

31. a treatment suffers the patient's of plasmodium infection method, it comprises the composition to the claim 21 of described patient's effective dosage.

32. the doubtful patient who contacts with plasmodium of treatment method, it comprises the composition to the claim 21 of described patient's effective dosage.

33. a method of preventing the malaria in the Mammals, it comprises the composition of the claim 21 of insect vector administration one tittle in carrying plasmodial insect vector colony.

34. the method for claim 33, wherein said peptide suppress the parasitemia of malaria in described patient's the HRBC of malaria infection.

35. the method for claim 31, wherein said plasmodium are selected from Plasmodium vivax (Plasmodium vivax) and plasmodium falciparum.

36. the method for claim 31, wherein said patient is infected by HIV in addition.

37. the method for claim 31, wherein said composition are with the second composition administration, described second composition comprises the activeconstituents that is selected from antimalarial drug and anti-HIV medicine.

38. the method for claim 37, the wherein composition of administration claim 21 within administration second composition 0 minute to 12 hours.

39. the method for claim 31, wherein oral by being selected from, suction, intravenously, intramuscular and hypodermic method are to the described composition of described patient's administration.

40. the method for claim 39, wherein through intravenously to the described composition of described patient's administration.

41. the method for claim 33, wherein oral administration is to the described composition of described insect vector administration.

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