CN101287502A - Compositions and methods for treating HIV infection with cupredoxin and cytochrame c - Google Patents

Abstract

The present invention relates to cupredoxin, specifically Pseudomonas aeruginosa azurin, and/or Pseudomonas aeruginosa cytochrome c551 and their use in inhibiting of viral infection, and in particular infection of mammalian cells by the Human Immunodeficiency Virus (HIV). The invention also relates to variants and derivatives of cupredoxin and cytochrome c that retain the ability to inhibit viral infection, and in particular infection by the Human Immunodeficiency Virus (HIV). The invention also relates to research methods for studying viral and bacterial infection in mammalian cells.

Description

Compositions and method with cupredoxin and cytochrome C treatment HIV infection

Related application

According to U.S.C the 119th joint and 120 joints, the application requires the U.S. Patent application No._______ that on May 18th, 2006, the common denomination of invention of submitting to was " Compositions and Methods for Treating Malariawith Cupredoxins and Cytochrome ", 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, the U.S. Patent application 11/244 that on October 6th, 833 and 2005 submitted to, 105 priority, 11/244,105 require the U.S. Provisional Patent Application 60/616 of submission on October 7th, 2004, the U.S. Provisional Patent Application 60/680 that on May 13rd, 782 and 2005 submitted to, 500 priority, and be the U.S. Patent application of submitting on November 11st, 2,003 10/720,603 part continuation application, 10/720,603 require the U.S. Provisional Patent Application 60/414 of submission on August 15th, 2003,550 priority, and be the U.S. Patent application of submitting on January 15th, 2,002 10/047,710 part continuation application, 10/047,710 require the priority of the U.S. Provisional Patent Application 60/269,133 of submission on February 15 calendar year 2001.Incorporate these full contents into the application by reference at this in first to file.

Government's rights and interests statement

The application's theme has been subjected to National Institutes of Health (NIH) (Bethesda, Maryland, the subsidy of research foundation U.S.A.) (fund number be AI 16790-21, ES 04050-16, AI45541, CA09432 and N01-CM97567).Government has some right in this invention.

Technical field

The present invention relates to the AZURIN. of cupredoxin (Cupredoxin), particularly Pseudomonas aeruginosa (Pseudomonas aeruginosa), and/or the Pseudomonas aeruginosa cytochrome C ₅₅₁, and their purposes in the infection of the mammalian cell that suppresses viral infection and particularly cause by human immunodeficiency virus (HIV).The invention still further relates to the variant and the derivant of cupredoxin and cytochrome C, they have kept the ability of the infection that inhibition viral infection, particularly human immunodeficiency virus (HIV) cause.The invention still further relates to the virus of research mammalian cell and the research method of bacterial infection.

Background technology

Human immunodeficiency virus (HIV) infection causes although this is a kind of infection new relatively among the mankind, has become the most important health problem in the whole world rapidly by AIDS.HIV/AIDS is the primary cause of the death on the south the African the Sahara now, and is the global the fourth-largest cause of the death.At the bottom of calendar year 2001, estimate that there are 40,000,000 HIV infection populations in the whole world.Center for Disease Control (CDC) (CDC) estimates that the U.S. has about 800,000 people to suffer from AIDS, and the case of 40,000 new diagnosis is arranged every year.Although now had some better Therapeutic Method can prolong the life of HIV infected patient, but still can not cure.

Modern inverase at be some different phases of HIV life cycle and HIV duplicates and the necessary some kinds of enzymes of surviving.Some inverases commonly used comprise nucleoside reverse transcriptase inhibitor such as AZT, ddI, ddC, d4T, 3TC and Abacavir (abacavir); Nucleotide reverse transcriptase inhibitors such as tenofovir (Tenofovir); Non-nucleoside reverse transcriptase inhibitor such as nevirapine (nevirapine), Yi Feiweilun (efavirenz) and Delavirdine (delavirdine); Protease inhibitor such as Saquinavir (saquinavir), ritonavir (ritonavir), indinavir (indinavir), viracept see nelfinaivr (nelfinavir), amprinavir, Lopinavir (lopinavir) and atazanavir (atazanavir); And fusion inhibitor such as En Fuwei peptide (enfuvirtide).But, at many HIV infected patients, no matter these antiviral drugs are separately or make up, all can not effectively prevent chronically infected process or be used for the treatment of acute AIDS.The high mutation rate of HIV virus and Drug resistance HIV strain therefore to occur be to cause effectively treating the big reason that HIV infects.Therefore need method new and that better treatment HIV infects.

Summary of the invention

The present invention relates to the compoistion and method of use of cupredoxin and/or cytochrome C 551 and be used to suppress the purposes that the viral infection of mammalian cell, particularly HIV infect.Particularly, the method for the growth infected of the HIV-1 that the present invention relates to comprise the compositions of peptide Pseudomonas aeruginosa AZURIN. (Azurin) and/or cytochrome C 551 and/or their variant and/or derivant and use these compositionss and peptide to suppress mammalian cell and people.The invention still further relates to the variant and the derivant of cupredoxin and cytochrome C 551, they have kept viral infection, the particularly ability of the growth of HIV infection of suppressing.

One aspect of the present invention is a kind of isolating peptide, and it is variant, derivant or the structural equivalents of cupredoxin or cytochrome C 551, and can suppress the growth that HIV-1 infects in the mammalian cell.In some embodiments, cupredoxin is AZURIN., false AZURIN. (Pseudoazurin), plastocyanin (Plastocyanin), Rusticyanin, Laz or Auracyanin, particularly AZURIN. or Laz.In other embodiments, cupredoxin is from Pseudomonas aeruginosa, alcaligenes faecalis (Alcaligenes faecalis), Achromobacter xylosoxidans (Achromobacter xylosoxidan), bordetella bronchiseptica (Bordetellabronchiseptica), methylomonas (Methylomonas sp.), Neisseria meningitidis (Neisseria meningitidis), Diplococcus gonorrhoeae (Neisseria gonorrhea), pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas chlororaphis (Pseudomonaschlororaphis), xyllela fastidiosa (Xylella fastidiosa), or vibrio parahaemolytious (Vibrioparahaemolyticus), particularly from Pseudomonas aeruginosa or Diplococcus gonorrhoeae.In some embodiments, described isolating peptide is the part of one of SEQ ID NO:1-17.In some embodiments, the sequence of described peptide and SEQ ID NO:1-17 has the aminoacid sequence homogeny at least about 90%.

In some embodiments, isolating peptide can be the clipped form of cupredoxin or cytochrome C 551.Described peptide can have about 10 residues of surpassing, but is no more than about 100 residues.Described isolating peptide comprises residue 36-128, the residue 36-88 of AZURIN. or the zone of residue 88-113.In other embodiments, described isolating peptide is made up of residue 36-128, the residue 36-88 of AZURIN. or the zone of residue 88-113.In other embodiments, described peptide comprises the residue of the cupredoxin that the zone with residue 36-128, the residue 36-88 of AZURIN. or residue 88-113 is equal to.

Another aspect of the present invention relates to a kind of compositions, it comprises at least a cupredoxin, cytochrome C 551 or the isolating peptide that is present in the pharmaceutical composition, variant, derivant or structural equivalents that described isolating peptide is cupredoxin or cytochrome C 551, and can suppress the growth that HIV-1 infects in the mammalian cell.In some embodiments, pharmaceutical composition is configured to and is used for intravenous and uses.In other embodiments, cupredoxin is from Pseudomonas aeruginosa, alcaligenes faecalis, Achromobacter xylosoxidans, bordetella bronchiseptica, methylomonas, Neisseria meningitidis, Diplococcus gonorrhoeae, pseudomonas fluorescens, Pseudomonas chlororaphis, xyllela fastidiosa or vibrio parahaemolytious, and particularly Pseudomonas aeruginosa or Diplococcus gonorrhoeae.In some embodiments, cupredoxin or cytochrome C 551 are selected from SEQID NO:1-17.

Another aspect of the present invention relates to the method for the treatment of the patient who has infected virus or antibacterial, comprise compositions to patient's administering therapeutic effective dose, described compositions comprises at least a cupredoxin, cytochrome C 551 or the isolating peptide that is present in the pharmaceutical composition, and described peptide is variant, derivant or the structural equivalents of cupredoxin or cytochrome C 551 and can suppresses the growth that HIV-1 infects in the mammalian cell.Described patient can infect HIV-1, herpes simplex virus (HSV), Ebola virus, cytomegalovirus (CMV), A, B and C type parainfluenza virus, A, B, C and G Hepatitis virus, hepatitis D virus (HDV), mumps virus, Measles virus, respiratory syncytial virus, Bunyavirus (bunyvirus), arenavirus, the upright virus of assistant (Dhori virus), poliovirus, rubella virus, dengue virus; SIV or mycobacterium tuberculosis, and HIV-1 particularly.In some embodiments, described patient is the people.In some embodiments, by intravenous injection, intramuscular injection, subcutaneous injection, suction, local application, transdermal patch, suppository and oral mode applying said compositions, particularly intravenous injection.Can be in 0 minute of the medicine of using another kind of anti-HIV to 1 week applying said compositions, particularly with the about while applying said compositions of another kind of antiviral, anti-bacterial drug and/or inverase.

Another aspect of the present invention relates to a kind of compositions, and it comprises variant, derivant or the structural equivalents of at least two kinds of isolating cupredoxins, cytochrome C 551 and cupredoxin or cytochrome C 551.In some embodiments, described compositions is present in the pharmaceutical composition.

Another aspect of the present invention relates to and comprises the test kit that is present in a kind of compositions in the bottle, described compositions comprises at least a cupredoxin, cytochrome C 551 or the isolating peptide that is present in the pharmaceutical composition, and the variant that described isolating peptide is cupredoxin or cytochrome C 551, derivant or structural equivalents third can suppress the growth that HIV-1 infects in the mammalian cell.In some embodiments, described test kit is used for intravenous and uses.

Another aspect of the present invention relates to the virus of studying mammalian cell and the method for bacterial infection, and its step comprises cell and cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents are contacted; Described cell is contacted with antibacterial or virus; And the growth of mensuration virus or bacterial infection.In some embodiments, cell and cupredoxin or cytochrome C 551 or the contacted step of its variant, derivant or structural equivalents were carried out before cell and antibacterial or viral contacted step.In other embodiments, cell and cupredoxin or cytochrome C 551 or the contacted step of its variant, derivant or structural equivalents are carried out after cell and antibacterial or viral contacted step.In some embodiments, cell is the human cell.In other embodiments, cell is lymphocyte or blood mononuclear cell on every side.In other embodiments, virus or antibacterial are HIV-1, herpes simplex virus (HSV), Ebola virus, cytomegalovirus (CMV), A, B and C type parainfluenza virus, A, B, C and G Hepatitis virus, hepatitis D virus (HDV), mumps virus, Measles virus, respiratory syncytial virus, Bunyavirus, arenavirus, the upright virus of assistant, poliovirus, rubella virus, dengue virus, SIV and mycobacterium tuberculosis, particularly HIV-1.

Another aspect of the present invention relates to expression vector, and its a kind of peptide of encoding, described peptide are variant, derivant or the structural equivalents of cupredoxin or cytochrome C 551 and can suppress the growth that HIV-1 infects in the mammalian cell.

By the explanation of following the drawings and specific embodiments, these and other aspects of the present invention, advantage and characteristics will be conspicuous.

The sequence explanation

SEQ ID NO:1: the aminoacid sequence of Pseudomonas aeruginosa AZURIN..

SEQ ID NO:2: from the aminoacid sequence of the cytochrome C 551 of Pseudomonas aeruginosa.

The aminoacid sequence of SEQ ID NO:3:Phormidium laminosum plastocyanin.

SEQ ID NO:4: the aminoacid sequence of thiobacillus ferrooxidant Rusticyanin.

The aminoacid sequence of the false AZURIN. of SEQ ID NO:5:Achromobacter cycloclastes.

SEQ ID NO:6: the aminoacid sequence of alcaligenes faecalis AZURIN..

SEQ ID NO:7: the aminoacid sequence of the AZURIN. of Achromobacter xylosoxidans denitrificans I.

SEQ ID NO:8: the aminoacid sequence of bordetella bronchiseptica AZURIN..

SEQ ID NO:9: the aminoacid sequence of methylomonas AZURIN..

SEQ ID NO:10: the aminoacid sequence of the AZURIN. of Neisseria meningitidis Z2491.

SEQ ID NO:11: the aminoacid sequence of pseudomonas fluorescens AZURIN..

SEQ ID NO:12: the aminoacid sequence of Pseudomonas chlororaphis AZURIN..

SEQ ID NO:13: the aminoacid sequence of the AZURIN. of xyllela fastidiosa 9a5c.

SEQ ID NO:14: the aminoacid sequence of the stellacyanin of Fructus Cucumidis sativi (Stellacyanin).

The aminoacid sequence of the Auracyanin A of SEQ ID NO:15:Chloroflexus aurantiacus.

The aminoacid sequence of the Auracyanin B of SEQ ID NO:16:Chloroflexus aurantiacus.

SEQ ID NO:17: the proteic aminoacid sequence of Fructus Cucumidis sativi alkalescence of Fructus Cucumidis sativi.

SEQ ID NO:18: the H.8 aminoacid sequence in district of the Laz of Diplococcus gonorrhoeae F62.

SEQ ID NO:19: the aminoacid sequence of the Laz of Diplococcus gonorrhoeae F62.

SEQ ID NO:20: the forward primer that is used for the Laz encoding gene (Laz) of pcr amplification Diplococcus gonorrhoeae.

SEQ ID NO:21: the reverse primer that is used for the Laz encoding gene (Laz) of pcr amplification Diplococcus gonorrhoeae.

SEQ ID NO:22: the segmental forward primer of 3.1kb that is used for pcr amplification pUC18-laz.

SEQ ID NO:23: the segmental reverse primer of 3.1kb that is used for pcr amplification pUC18-laz.

SEQ ID NO:24: the segmental forward primer of 0.4kb that is used for pcr amplification pUC19-laz.

SEQ ID NO:25: the segmental reverse primer of 0.4kb that is used for pcr amplification pUC19-laz.

SEQ ID NO:26: the forward primer that is used for pGST-azu 36-128.

SEQ ID NO:27: the reverse primer that is used for pGST-azu 36-128.

SEQ ID NO:28: the forward primer that is used for pGST-azu 36-89.

SEQ ID NO:29: the reverse primer that is used for pGST-azu 36-89.

SEQ ID NO:30: the forward primer that is used for pGST-azu 88-113.

SEQ ID NO:31: the reverse primer that is used for pGST-azu 88-113.

SEQ ID NO:32: be used for the oligonucleotide of direct mutagenesis with preparation pGST-azu 88-113.

SEQ ID NO:33: be used for the oligonucleotide of direct mutagenesis with preparation pGST-azu 88-113.

Description of drawings

Fig. 1: Fig. 1 illustrates AZURIN., H.8-AZURIN. (H.8-Az) and Laz suppress the growth of HIV-1 virus.These protein are hatched with variable concentrations and PBMC, add the HIV-1 of 3 kinds of hypotypes subsequently.After hatching 2h, remove virus, but as described in embodiment 3, protein is added.Determine the inhibition of viral growth by elisa assay p24.

Fig. 2: Fig. 2 illustrates the surface plasma resonance binding curve, and this curve illustrates the binding pattern of cupredoxin and CD4 and HIV-1 gp120.(A) the SPR titration curve shows that AZURIN. and GST-Azu 36-128 (illustrating with illustration) combine with the new specificity of immobilized CD4 (CD4-CM5) on the golden sensor chip of carboxymethyldextran bag quilt.HIV-1gp120, HIV-1gag and HIV-1 nef are respectively as the positive and negative control.By with data fitting in R _Cq=R _Max/ (1+ (K _d/ C)), curve fitting connects above-mentioned data point, determines RA thus.CD4 is in conjunction with K _dValue is: 36.9 ± 2.0nM (AZURIN .), 0.34 ± 0.04nM (GST-Azu 36-128) and 48.1 ± 3.1nM (HIV-1 gp120).(B) when immobilized AZURIN. (Az-CM5) contacts with HIV albumen in conjunction with titration.Because naked Au-CM5 chip has a large amount of non-specific binding, therefore CM5 is joined in the electrophoretic buffer as eluent and (CM5 is added in the HBS-EP buffer) according to 1mg/ml.The Kd that curve fitting obtains is 25.1 ± 3.1nM (CD4) and 8.9 ± 0.8nM (HIV-1 gp120).(C) by under the condition similar, determining ICAM (ICAM-1, ICAM-2, ICAM-3 and NCAM, illustration) and the bonded SPR curve of immobilized AZURIN. to the experiment of (B) part.The selectivity identification of AZURIN. and ICAM-3 but not ICAM-I or ICAM-2 is very remarkable, and bond strength is 19.5 ± 5.4nM.The bonded Kd of NCAM and AZURIN. shown in illustration, is 20 ± 5.0nM.(D) carry out SPR in conjunction with competition research with the CD4 that is immobilized onto on the CM5 sensor chip.AZURIN .+HIV-1 gp120 solution is added to sensor surface, the concentration difference of AZURIN. (AZURIN.: 0-4500nM, HIV-1 gp120:242nM) wherein, with the ratio of data as resonance, i.e. the % of overall reaction, [R maps _Eq(AZURIN .+HIV-1 gp120)/(R _Eq/ (HIV-1 gp120))].Use HIV-1 gp120, carry out the titration of GST-Azu36-128, and analyze in a similar fashion immobilized CD4.Competition Notes of Key Data AZURIN. and GST-Azu 36-128 are 1: 1 with the stoichiometry that combines between the immobilized CD4.

Fig. 3: Fig. 3 illustrates surface plasma resonance in conjunction with titration, and it shows the interaction of AZURIN. and GST-AZURIN. fusant and DC-SIGN.(A) be injected to the CM5 sensor surface that DC-SIGN modifies by protein (0-100nM) and determine that AZURIN., ICAM-3 and GST-Azu 36-89 combine with the concentration dependent of DC-SIGN, and (resonance units: function RU) is estimated as balance resonant reaction value with combination degree variable concentrations.(B) GST-Azu 88-113 and DC-SIGN's has adopted as the A part with regard to described identical chip of AZURIN. and scheme in conjunction with titration curve.The positive of GST-Azu 88-113 and DC-SIGN interact prompting its have latent effect as the recognition sequence of AZURIN..By with data fitting to R _Eq=R _Max/ (1+ (Kd/C)) and determine binding affinity (Kd) to AZURIN., ICAM-3 and GST-Azu88-113 and the data point in the curve fitting connection layout.The Kd value of extrapolating is 0.83 ± 0.05nM (AZURIN .), 0.93 ± 0.39nM (ICAM-3) and 5.98 ± 1.13nM (GST-Azu 88-113).

Detailed Description Of The Invention

Definition

Unless distinguishingly be described as " individual cells ", term " cell " this comprise odd number or The plural number term.

Term " polypeptide ", " peptide " and " albumen " can Alternates at this, and it refers to amino acid The polymer of residue. This term is applicable to that wherein one or more amino acid residues are corresponding natural The amino acid polymer that has the amino acid residue of amino acid whose artificial chemistry analog. This term Also be applicable to the natural amino acid polymer that exists. Term " polypeptide ", " peptide " and " albumen " also comprise Modify, include but not limited to glycosylation, lipid connection, sulphation, glutaminic acid residue the γ carboxylation, Carboxylation and ADP ribosylation. Be appreciated that polypeptide is not all is fully linear. For example, Because the result of ubiquitin, polypeptide can be branch, and they can be annular (have or Do not have branch), all is the result of post-translational events generally, comprise natural process event and can the sky The event that the right artificial treatment that takes place is brought. By the natural process of untranslated and by complete Synthetic method can be synthesized annular, branch and polypeptide branch's annular.

Term " pathologic pathology " comprises at this and is different from normal dissection and physiological deviation, It has consisted of living animal or the wherein infringement of normal condition of a part, interrupts or has modified The performance of somatic function, and be a kind of to various factors (for example malnutritive, industrial hazard or Weather), to special infector (for example worm, parasitic protozoa, bacterium or virus), to organism Birth defect (for example genetic alteration) or the replying of the combination of these factors.

Term " pathology " comprises at this and is different from normal dissection and physiological deviation, its formation To living animal or the wherein infringement of normal condition of a part, interrupt or modified the body merit The performance of energy.

Term " suffers from " at this and comprises the symptom that shows at present the pathologic pathology, has even do not have Have observable symptom the pathologic pathology, be in convalescence of pathologic pathology and Rehabilitation from the pathologic pathology.

Term " treatment " comprises prevention, slows down, stops or reverses and the pathology phase for the treatment of at this The pathology of closing or progress or the severity of symptom. Therefore, in situation about working as, term " treatment " Comprise medical treatment, curative and/or preventative using.

Term " suppresses the growth that HIV infects " and refers to reduce the HIV infection of human body or prevent at this End any means of its increase. These means include but not limited to suppress HIV and genomicly copy, Suppress the synthetic and/or assembling of HTV capsid protein and suppress HIV to enter do not infect thin Born of the same parents. This definition comprises that any current known HIV treats any method of measure. Determine HIV Infect whether repressed a kind of method can be referring to embodiment 3.

" treatment effective dose " is the showing of special pathology that can effectively stop or slow down institute's treatment target Symptomatic progress or partially or completely eliminate the dosage of described symptom. Determine that the treatment effective dose is Within those skilled in the art's limit of power.

When be used for modifying term polypeptide or other compounds, term " basically purifying " exists This refers to a peptide species or compound, for example isolates from growth medium or cell component Polypeptide, its form is not for basically having or do not mix other albumen and/or active inhibitionization Compound. Term " basically purifying " refers to, with dry weight basis, and the branch of the amount of the part of separation Number is at least about 75%, or at least " 75% basically purifying ". More specifically, term is " basic Upper purifying " amount of referring to be at least about the reactive compound dry weight 85% or " 85% is basic at least Upper purifying " compound. The most concrete, term " basically purifying " amount of referring to is at least about 95% or " 95% basically purifying " compound at least of reactive compound dry weight. Basically described The cupredoxin of purifying or Cytochrome C551 or its variant or derivative can be united a kind of Or multiple other basically compound or another kind of cupredoxin or cell looks that separate of purifying Plain C551 uses.

Phrase " separation ", " purifying " or " the biology purifying " refer to, and a kind of material is basic Upper or do not contain in fact those are accompanied by described material usually in native state component. Therefore, The peptide of separation of the present invention preferably is not contained in the in situ environment of peptide and the normal relevant thing of peptide Matter. " separation " district refers to the zone of the whole sequence that does not contain the polypeptide of originating in this zone. " divide From " nucleic acid, albumen or its respective segments shift out from its vivo environment basically, so that It can be processed processing by those skilled in the art, such as but not limited to nucleotide sequencing, restricted disappearing Change, directed mutagenesis and nucleic acid fragment by subclone in expression vector and obtain basically The albumen of purifying quality or protein fragments.

When relating to peptide, term " variant " referred to herein as the amino acid sequence variant, and it is with wild The type of giving birth to polypeptide is compared and can be had amino acid replacement, disappearance or that insert. Variant can It is the clipped form of wild type peptide. Therefore, can prepare by the gene of processing coded polypeptide Variant peptides. By basic composition or the feature of change polypeptide, but do not change at least some its bases This activity can be prepared variant. For example, " variant " of Bazurin can be the sky of sudden change Green white, it has kept the ability of the growth of the HIV infection that suppresses in the mammal. At some In the situation, with alpha-non-natural amino acid for example ε-(3,5-dinitrobenzoyl)-the synthetic variant of Lys residue Peptide (Ghadiri ﹠ Fernholz, J.Am.Chem.Soc, 112:9633-9635 (1990)). One In a little embodiments, compare with wild type peptide, variant can have be no more than 20 replacements, Amino acid disappearance or that insert. In some embodiments, compare with wild type peptide, become Body can have the amino acid that is no more than 15 replacements, disappearance or insertion. In some enforcement In the mode, compare with wild type peptide, variant can have be no more than 10 replacements, the disappearance Or the amino acid that inserts. In some embodiments, compare with wild type peptide, variant can Be no more than amino acid 6 replacements, disappearance or that insert to have. At some embodiments In, compare with wild type peptide, variant can have be no more than 5 replacements, the disappearance or The amino acid that inserts. In some embodiments, compare with wild type peptide, variant can tool Have and be no more than amino acid 3 replacements, disappearance or that insert.

Term " amino acid " comprises that arbitrary naturally occurring or non-natural exists or closes in this expression The amino acid moiety of the amino acid residue that becomes, namely comprise through 1,2,3 or more carbon atom (logical Often be 1 (α) carbon atom) the appointing of direct-connected at least one carboxyl and at least one amino What part.

When relating to peptide, term " derivative " referred to herein as and is derived from object peptide (subject Peptide) peptide. Derivatization comprises the chemical modification peptide, and it is basic so that peptide still keeps some Active. For example, " derivative " of Bazurin can be to keep it to suppress HIV in the mammal The Bazurin of the chemical modification of the ability of the growth of infecting. Interested chemical modification comprise but Amidatioon, acetylation, sulphation, the polyethylene glycol (PEG) that is not limited to peptide modified, phosphorylation Or glycosylation. In addition, derived peptide can be the fusion of polypeptide or its fragment and compound, and is described Compound is such as but not limited to another kind of peptide, drug molecule or other treatment or pharmaceutical agent But or detector probe.

Term " amino acid sequence homogeny percentage (%) " is defined as being compared when two sequences The time, the percentage of the amino acid residue identical with amino acid residue in the candidate sequence in the polypeptide. In order to determine amino acid homogeny %, aligned sequences if necessary, is then introduced breach, so that Obtain maximum sequence homogeny %, conservative replacement is not considered to the part of sequence homogeny. The amino acid sequence comparison method of determining homogeny percentage is that those skilled in the art are known. Use Computer software that usually can be public is BLAST, BLAST2, ALIGN2 or Megalign for example (DNASTAR) software comparison peptide sequence. In a concrete embodiment, use Blastp (from state-run biotechnology information centre, Bethesda MD), it uses long complexity filter The default parameter of (long complexity filter), expection (expect) 10, word length (word size) 3, there be (existence) 11 and extend 1.

When comparison during amino acid sequence, given amino acid sequence A with or with respect to the amino acid sequence homogeny % of given amino acid sequence B (itself or can be expressed as given amino acid sequence A have comprise with or with respect to the specific amino acid sequence homogeny % of given amino acid sequence B) can be calculated as:

Amino acid sequence homogeny %=X/Y*100

Wherein

X is by the sequence alignment program of A and B or the comparison of algorithm are integrated to identical The number of the amino acid residue of coupling; And

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

If the sequence of the length of amino acid sequence A and amino acid sequence B is unequal, so A To be not equal to the amino acid sequence homogeny % of B and A with the amino acid sequence homogeny % of B. When relatively long sequence during with short sequence, short sequence will be " B " sequence. For example, When the peptide that compares clipped form and corresponding wild type peptide, the peptide of clipped form will be " B " order Row.

General introduction

The present invention relates to comprise composition and the inhibition of cupredoxin and/or Cytochrome C551 The method that the virus of mammalian cell and mammalian subject and bacterium infect. The present invention is concrete Relate to the composition that comprises cupredoxin and/or Cytochrome C551 and suppressing people's immunity Purposes in the growth that defective virus (HIV) infects. The invention still further relates to cupredoxin and thin The variant of Cytochrome C 551, derivative and structural equivalents, they have kept the inhibition virus infections And the ability of the growth infected of HIV particularly, and relate to the composition that comprises above-mentioned substance. Particularly, the invention provides composition, it comprises Azurin and cell look Variant, derivative and the structural equivalents of plain C551, Bazurin and Cytochrome C551, And be used for the treatment of and suffer from that virus or bacterium infect and the patient's that infects of HIV-1 usefulness particularly On the way, or its be used in the pre-aseptic purposes of the people with risk of infection. At last, the present invention Be provided for studying mammalian cell by the method for virus or bacterium infection, it passes through at cell Infected before or afterwards, with cell and cupredoxin or Cytochrome C551 or its change Body, derivative or structural equivalents contact, and measure the growth of infecting.

Two kinds of before this known, that pseudomonas aeruginosa generates redox proteins, i.e. cupredoxin Bazurin (Azurin) and cromoci₅₅₁(Cyt C ₅₅₁), it is thin that both can both enter J774 Born of the same parents, and compare with normal cell, it has demonstrated significant cell to the human cancer cell Cytotoxic activity (Zaborina et al, Microbiology 146:2521-2530 (2000)). Bazurin Also can selectively enter Humanmachine tumour UISO-Mel-2 or MCF-7 Human Breast Cancer Cells (Yamada et al, PNAS 99:14098-14103 (2002); Punj et al, Oncogene 23:2367-2378 (2004); Yamada et al, Cell.Biol.7:14181431 (2005)). From The Bazurin of pseudomonas aeruginosa preferably enters J774 mouse reticulosarcoma cell, with Tumor suppressor protein p53 forms compound and stablizes p53 albumen, has increased in the cell of p53 Concentration, and apoptosis-induced (Yamada et al, Infection and Immunity, 70:7054-7062 (2002)).

Amazing is to learn now around blood mononuclear cell of Bazurin (PBMC) induce about 90% HIV-1 growth inhibition in the culture. See embodiment 3. Now Learn that Bazurin suppresses the growth of three kinds of HIV-1 strains, namely Bal (is popular in the U.S. and West Europe Topmost clade B), clade B Africa separated strain RW/92/008/RE1 and advancing Change branch C India separated strain IN/2167 D15. See embodiment 3. In addition, also learn now from The cupredoxin sample albumen of Neisseria, namely Laz suppresses the growth of these three kinds of HIV-1 strains, H.8 the district of Laz albumen also is like this with the fusion of Azurin. See enforcement Example 3. At last, learn now Bazurin and Cytochrome C551 from pseudomonas aeruginosa The M44KM64E mutant can suppress HIV sense in the Human Lymphocytes that HIV infects Dye. See embodiment 1.

Learn that now the Bazurin of pseudomonas aeruginosa and ICAM-I (see HIV-1 particle and Know the infectivity that can increase HIV-1), ICAM-2 and CD4 (the primary cell table of HIV-1 The face acceptor) has structural similarity. See embodiment 2. Surface plasma resonance is tested and is had now found that, Bazurin demonstrates obviously in conjunction with cell surface receptor CD4 external, and beyond expectation is, Its binding affinity to CD4 is higher than HIV-1 surface ligand gp120. See embodiment 4. This Outward, GST-Azu 36-128, i.e. glutathione S-transferase (GST) and Bazurin amino acid The fusion of 36-128, stronger than Bazurin itself with the combination of CD4, and GST-Azu 88-113 is not in conjunction with CD4, and prompting only is that the part of Bazurin is responsible in conjunction with CD4. See Embodiment 4. The combination of Gp120 and Bazurin is stronger with the combination of CD4, and the sky is described Green white both in conjunction with gp120 also in conjunction with CD4. In addition, in surface plasma resonance is analyzed, ICAM-3 and NCAM demonstrate strongly in conjunction with Bazurin external. See embodiment 5. After, now learn Bazurin in conjunction with another kind of HIV-1 acceptor, namely specific for dendritic cells is sticking Attached acceptor (DC-SIGN). See embodiment 7. Participate in other eggs that HIV-1 enters host cell White matter is gp41 for example, then with Bazurin any combination does not take place. See embodiment 4.

By competitive assay, learn that now Bazurin is of the same race with it at the external gp120 that can disturb The combination of acceptor CD4. Particularly, GST-Azu 36-128 demonstrates and gp120 competition combination The remarkable ability of CD4, GST-Azu 88-113 does not then almost have competitiveness. See embodiment 6. Laz is a kind of Bazurin sample protein from Neisseria, if existing known infected by HIV-Added before 1, it can suppress the growth of HIV-1 infection in the PBMC culture and reach 73%, And if after infected by HIV-1, add then almost not inhibition. See embodiment 8. Although do not have Be intended to running of the present invention is limited to any mechanism, but seem that Bazurin can lead to Cross disturb HIV and cell surface receptor for example between ICAM, CD4 and the DC-SIGN mutually Effect and prevent that HIV from entering cell and infection cell is infected around blood thereby suppress HIV-1 Growth in the mononuclearcell.

Therefore, owing to have the structural similarity of height between the cupredoxin, so other copper Oxygen also albumen also can suppress the growth of HIV-1 infection in the mammal haemocyte. Therefore, remove Beyond HIV infects, cupredoxin also can suppress those in conjunction with and ICAM, DC-SIGN Have other Viral infections of the cell surface receptor of similar structures with CD4. For example, The bind receptor of DC-SIGN or other virus or bacterial pathogens, these pathogen comprise third Hepatitis virus (HCV), Ebola virus, cytomegalovirus (CMV) and tuberculosis branch bar Bacterium (Wang et al, Chin.Med.J.117:1395-1400 (2004)).

Composition of the present invention

Peptide provided by the invention is cupredoxin or pseudomonas aeruginosa Cytochrome C551 Variant, derivative or structural equivalents. In the following embodiments, described peptide is basically pure . In other embodiments, described peptide is present in the composition, and said composition comprises described Peptide or basically formed by described peptide. In other embodiments, peptide separates. One In a little embodiments, peptide is shorter than total length cupredoxin or Cytochrome C551, and it has kept copper Oxygen is some functional characters of albumen or Cytochrome C551 also. In some embodiments, peptide is protected Stayed in the inhibition Peripheral blood mononuclearcell virus or bacterium to infect and HIV-1 more particularly The ability of the growth of infecting. In concrete embodiment, Cytochrome C551 is SEQ ID NO:2. In another concrete embodiment, peptide can not cause mammiferous immune response, People's immune response more particularly. The present invention also provides composition, and it comprises at least a peptide, Described peptide be cupredoxin, pseudomonas aeruginosa Cytochrome C551 or cupredoxin or The variant of Cytochrome C551, derivative or structural equivalents. The present invention also provides and comprises existence The composition of at least a peptide in pharmaceutical composition, described peptide are cupredoxin, verdigris The variant of pseudomonad cells pigment C551 or cupredoxin or Cytochrome C551, spread out Biology or structural equivalents.

Because the attach structure homology between the cupredoxin, expect other cupredoxins with Azurin is the same, also has phase aspect the growth that suppresses the HIV-1 infection HIV-resistant activity together. In some embodiments, cupredoxin is but is not limited to reddish black egg In vain, false Bazurin, plastocyanin, Rusticyanin or Auracyanin. Special at some In the concrete embodiment, Bazurin is derived from pseudomonas aeruginosa, Alcaligenes faecalis, oxidation wood The denitriflcans I, bordetella bronchiseptica, methylomonas of sugar achromobacter, Neisseria meningitidis Z2491, NEISSERIA GONORRHOEAE, Pseudomonas fluorescens, Pseudomonas chlororaphis, severe Support rod bacterium 9a5 or vibrio parahaemolytious. In very concrete embodiment, Bazurin comes From pseudomonas aeruginosa. In other concrete embodiments, cupredoxin comprises amino acid Sequence SEQ ID NO:1,3-17. In other specific embodiment, cupredoxin be from The Laz albumen of Neisseria meningitidis or NEISSERIA GONORRHOEAE.

Invention provides the amino acid sequence variant of cupredoxin or Cytochrome C551, its with Wild type peptide is compared has amino acid replacement, disappearance or that insert. Variant of the present invention It can be the clipped form of wild type peptide. In some embodiments, composition comprises by little The peptide that forms in the zone of the cupredoxin of total length wild type peptide or Cytochrome C551. In some embodiments, composition comprises by the cupredoxin of clipped form or cytochromes Surpassing about 10 residues, surpass about 15 residues or surpassing about 20 residues of C551 forms Peptide. In some embodiments, composition comprises by the cupredoxin of clipped form or thin Being no more than about 100 residues, being no more than about 50 residues, being no more than approximately of Cytochrome C 551 40 residues or be no more than the peptide that about 30 residues form. In some embodiments, composition Comprise with cupredoxin or Cytochrome C551 and more specifically with SEQ ID NO:1-17 has at least about 90% amino acid sequence homogeny, at least about 95% amino acid sequence Homogeny or at least about the peptide of 99% amino acid sequence homogeny.

In concrete embodiment, the variant of cupredoxin comprises the sky of pseudomonas aeruginosa Green white residue 36-128, Bazurin residue 36-88 or Bazurin residue 88-113. In other embodiments, the variant of cupredoxin is by the Bazurin residue of pseudomonas aeruginosa 36-128, Bazurin residue 36-88 or Bazurin residue 88-113 form. At other In the specific embodiment, variant is by the residue group of equal value of the cupredoxin except Bazurin Become. Expect that also the cupredoxin variant can be designed to and Bazurin residue 36-128, reddish black Protein residues 36-88 or Bazurin residue 88-113 have similar activity. For this reason, use BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), to be positioned at verdigris false single Can on relevant residue on the born of the same parents bacterium Bazurin amino acid sequence and the object cupredoxin sequence The clipped form variant that is equal to that is equal to residue and therefore designs of seeing is compared object copper oxygen Also Argine Monohydrochloride sequence and Azurin sequence.

Variant also includes the peptide that synthesizing amino acid that non-natural exists is made. For example, non-natural The amino acid that exists can be integrated in the variant peptides, so that extension or optimum organization thing are at blood flow In half-life. These variants include but not limited to D, L-peptide (diastereomer) (Futaki et 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)), contain rare amino acid whose peptide (Lee et al., J.Pept.Res.63 (2): 69-84 (2004)) and the alpha-non-natural amino acid that contains alkene continue with hydrocarbon lock ring (stapling) (Schafmeister et al, J.Am.Chem.Soc.122:5891-5892 (2000); Walenski et Ah, Science 305:1466-1470 (2004)) and comprise ε-(3,5-dinitrobenzoyl)-Lys The peptide of residue.

In other embodiments, peptide of the present invention is cupredoxin or Cytochrome C551 Derivative. The derivative of cupredoxin and/or Cytochrome C551 is that the chemistry of described peptide is repaiied The decorations form is so that peptide still keeps some its primary activities. " derivative " of Bazurin for example Can be the Bazurin of chemical modification, it has kept, and HIV infects in the inhibition mammalian cell The ability of growth. Interested chemical modification includes but not limited to, the amidatioon of peptide, acetyl Change, sulphation, polyethylene glycol (PEG) modification, phosphorylation and glycosylation. In addition, derived peptide Can be cupredoxin or Cytochrome C551 or its variant, derivative or structural equivalents With the fusion of compound, described compound is such as but not limited to another peptide, drug molecule Or but other treatment or medicine are with reagent or detector probe. Interested derivative comprise by It can prolong or optimize peptide of the present invention and the chemistry of the half-life of composition in blood flow is repaiied Decorations for example by the known method of some those skilled in the art, include but not limited to the cyclisation peptide (Circularized peptide) (Monk et al, BioDrugs 19 (4): 261-78, (2005); DeFreest et al, J.Pept.Res.63 (5): 409-19 (2004)), N-and C-are end modified (Labrie et al, Clin.Invest.Med.13 (5): 275-8, (1990)) and contain the non-natural of alkene Amino acid continues with hydrocarbon lock ring (Schafmeister et al., J.Am.Chem.Soc. 122:5891-5892 (2000); Walenski et al., Science 305:1466-1470 (2004)).

Expect peptide of the present invention can be cupredoxin or Cytochrome C551 variant, derive Thing and/or structural equivalents. For example, peptide can be by the brachymemma of the Bazurin of PEGization Therefore form is so that it is variant simultaneously is again derivative. In one embodiment, use Contain the α of the tethers (tether) with alkene, the disubstituted alpha-non-natural amino acid of α synthesizes this Bright peptide, and the subsequently olefin metathesis effect by ruthenium catalysis forms full hydrocarbon " lock Ring " (Scharmeister et al, J.Am.Chem.Soc.122:5891-5892 (2000); Walensky et al, Science 305:1466-1470 (2004)). In addition, as Bazurin The peptide of structural equivalents can merge with other peptides, therefore prepare be simultaneously structural equivalents and The peptide of derivative. These embodiment are just in order to illustrate the present invention, rather than restriction the present invention. The variant of cupredoxin or Cytochrome C551, derivative or structural equivalents can in conjunction with or Can be not in conjunction with copper.

In another embodiment, peptide is the structure of cupredoxin or Cytochrome C551 etc. The valency thing. The research of determining the significant structural homology between cupredoxin and other peptides is real Example comprises Toth et al. (Developmental Cell 1:82-92 (2001)). Particularly, by making Determine significant structure homology between cupredoxin and the structural equivalents with the VAST algorithm Property (Gibrat et al, Curr Opin Struct Biol 6:377-385 (1996); Madej et al, Proteins 23:356-3690 (1995)). In concrete embodiment, from cupredoxin With the structure of structural equivalents VAST p value relatively less than about 10^-3, less than about 10^-5, less than about 10^-7 In other embodiments, by use the DALI algorithm determine cupredoxin and Significant structural homology between the structural equivalents (Holm ﹠ Sander, J.MoI.Biol. 233:123-138 (1993)). In concrete embodiment, pairing structure DALI Z relatively Integration is at least about 3.5, at least about 7.0 or at least about 10.0.

In some embodiments, cupredoxin or Cytochrome C551 or its variant, spread out Biology or structural equivalents have one of Azurin or Cytochrome C551 A little functional characters. In a concrete embodiment, cupredoxin or Cytochrome C551 Suppress in the mammalian cell, more specifically for to have infected in the Peripheral blood mononuclearcell of HIV Virus or bacterium infect, the particularly growth of HIV virus infections. The present invention also provides copper oxygen also The variant of albumen and Cytochrome C551, derivative and structural equivalents, they have kept inhibition The growth that virus or bacterium infection, particularly HIV infect in the mammalian cell divides ability. Logical Cross commercial p24 enzyme immunoassay (EIA) (PerkinElmer Life Sciences, Inc., Wellseley, Mass.), by measuring the generation of HIV-1 p24 antigen in the cell culture supernatant, can determine The growth that HIV-1 infects in the cell. The inhibition of infecting growth is for the control treatment group Have any minimizing of infection of significance,statistical or the reduction that infection is advanced the speed.

Because known cupredoxin and Cytochrome C551 can suppress virus or bacterium infects, the spy Not the growth of the infection in the mammalian cell that has infected HIV, therefore can establish now Count out variant and the derivative of cupredoxin, their to have kept this antiviral or antibacterium, The activity of anti-HIV particularly. By for example utilizing a kind of in the several different methods known in the art Method generates cupredoxin and the various variants of Cytochrome C551 and " library " of derivative, Test then antiviral or antibacterial activity, the particularly HIV-resistant activity of every kind of material, just can To prepare this type of variant and derivative, for example adopt the exemplary method of embodiment 3. Expection institute The change of the cupredoxin with antiviral or antibacterium and particularly HIV-resistant activity that forms Body and derivative can be used to method of the present invention, to substitute cupredoxin or cytochromes C551 or use common with it.

In some concrete embodiments, cupredoxin or cytochromes or its variant, Derivative or structural equivalents are in conjunction with CD4, and its binding constant is different from non-binding statistically Reference protein. Can come test peptides with surface plasma resonance analysis as described in Example 4 This activity. Determine that whether a kind of protein be ability in conjunction with the additive method of another kind of protein The territory is known, also can use.

In some concrete embodiments, cupredoxin or cytochromes or its variant, Derivative or structural equivalents are in conjunction with gp120, and its binding constant is different from non-binding statistically Reference protein. Can come test peptides with surface plasma resonance analysis as described in Example 4 This activity. Determine that whether a kind of protein be ability in conjunction with the additive method of another kind of protein The territory is known, also can use.

In some concrete embodiments, cupredoxin or cytochromes or its variant, Derivative or structural equivalents are in conjunction with ICAM3, and its binding constant is different from non-knot statistically Close reference protein. Can be with surveying such as embodiment 4 and 5 described surface plasma resonance analyses This activity of examination peptide. Determine that a kind of protein is whether in conjunction with the additive method of another kind of protein Be known in the art, also can use.

In some concrete embodiments, cupredoxin or cytochromes or its variant, Derivative or structural equivalents are in conjunction with DC-SIGN, and its binding constant is different from non-statistically In conjunction with reference protein. Can be with coming such as embodiment 4 and 5 described surface plasma resonance analyses This activity of test peptides. Determine that a kind of protein is whether in conjunction with its other party of another kind of protein Method is known in the art, also can use.

In some concrete embodiments, inducing peptide mammalian cancer cells of the present invention (has more Body be the J774 cell) apoptosis. By utilizing MITOSENSOR^TMAPOLERT ^TMMitochondrial membrane sensor reagent box (Clontech Laboratories, Inc., Palo Alto, California, U.S.A.) Mitosensor ApoAlert Laser Scanning Confocal Microscope is by measuring caspase-8, caspase-9 and caspase-3 activity and by utilizing for example APOLERT with the described method such as Zou (J.Biol.Chem.274:11549-11556 (1999))^TMThe dna fragmentation kit (Clontech Laboratories, Inc., Palo Alto, California, U.S.A.) detects apoptosis induction The nuclear dna fragmentationization can observe the ability of cupredoxin or other polypeptid induction apoptosis.

In another concrete embodiment, the cell growth retardation of inducing peptide mammalian cancer cells of the present invention (more particularly J774 cell).By for example determining the cell growth retardation with the inhibition degree of described method mensuration such as Yamada (PNAS101:4770-4775 (2004)) cell cycle process.In another concrete embodiment, cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents have suppressed the cell cycle progression of mammalian cancer cells (more particularly J774 cell).

The cupredoxin class

These little blue copper proteins (cupredoxin) are to participate in the electron transfer protein (10-20kDa) of antibacterial electron transport chain or the albumen of unknown function.Copper ion is alone by the combination of albumen substrate institute.The planar delta of two histidine around the copper and the idio-morphosis of a cysteine part is arranged very peculiar Electronic Performance and the intensive blueness that has provided the metal site.Characterized out a large amount of cupredoxins to high-resolution ground crystallography.

Cupredoxin generally all has low sequence homology, but has high structural homology (Gough ﹠amp; Clothia, Structure 12:917-925 (2004); De Rienzo et al., Protein Science 9:1439-1454 (2000)).For example, the aminoacid sequence of the aminoacid sequence of AZURIN. and Auracyanin B has 31% homogeny, with the aminoacid sequence of plastocyanin 20.3% homogeny is arranged, and with the aminoacid sequence of false AZURIN. 17.3% homogeny is arranged.See Table 1.But these proteic structural similarities are more significant.The VAST p value of the structure comparison of AZURIN. and Auracyanin B is 10 ^-7.4, AZURIN. and Rusticyanin are 10 ^-5, AZURIN. and plastocyanin are 10 ^-5.6, and AZURIN. and false AZURIN. are 10 ^-4.1

All cupredoxins all have Greece's lace β bucket of 8 chains or the site structure (De Rienzo et al, Protein Science 9:1439-1454 (2000)) that the β sandwich folds and has high conservative.Exist around AZURIN. class, Amicyanin class, basket antibacterial plastocyanin class, the proteic copper of Fructus Cucumidis sativi alkalescence site owing to have a plurality of long-chain fat family residue for example methionine and the formed obvious hydrophobic sticking patch of leucine (hydrophobic patch), the blue plain class of false AZURIN. and plastid also has the hydrophobicity sticking patch of less degree.Go out the hydrophobicity sticking patch that also can find less degree at stellacyanin and Rusticyanin copper site, but have different performances.

Table 1: utilize the VAST algorithm to Pseudomonas aeruginosa AZURIN. (1JZG) and other proteic sequences and structure comparison.

¹Comparison length: the C-alpha atom of eclipsed equivalence is to number between two structures, and promptly to be used to calculate 3D overlapping for how many residues.

²P-VAL:VAST p value is the measured value of comparison significance, expressing possibility property.For example, if the p value is 0.001, see that by pure chance opportunity the paired probability of this quality is 1000: 1 so.For the effect of a plurality of comparisons, utilize supposition promptly in the MMDB data base, to have 500 independently to adjust VAST p value with the domain of irrelevant type.Shown p value therefore corresponding to the p value of the right paired comparison of each domain divided by 500.

³Integration: VAST structural similarity integration.This numeral is relevant with the number and this eclipsed quality of eclipsed secondary structure element.Higher VAST integration has higher similarity.

⁴RMSD: the overlapping residue of root root-mean-square (dust).After the optimization of two structures is overlapping, calculate this numerical value with the square root of the variance distance between the C-alpha atom of equal value.Notice that the RMSD numerical scale is relevant with the degree of structure comparison,, must consider this size as usefulness RMSD during as the description index of population structure similarity.

⁵Caenorhabditis elegans: confirmation is the main sperm protein (Kuwabara of the Ephrin antagonist in the oocyte maturation, 2003 " The multifaceted C.elegans major spermprotein:an Ephrin signalling antagonist in oocyte maturation " Genes andDevelopment, 17:155-161).

AZURIN.

AZURIN. is the cuprein that contains of 128 amino acid residues, and it belongs to the cupredoxin family of the electron transport that participates in specific bacteria.AZURIN. comprises those AZURIN. from Pseudomonas aeruginosa (PA) (SEQ ID NO:1), Achromobacter xylosoxidans and A.Denitrificans (Murphy etah, J.MoI.Biol.315:859-871 (2002)).Aminoacid sequence homogeny between the AZURIN. changes between 60-90%, and these albumen have demonstrated strong structural homology.All AZURIN. all have distinctive β-sandwich and Greece's lace motif, and single copper atom all is positioned at proteic same area all the time.In addition, AZURIN. has the neutral substantially hydrophobicity sticking patch that is centered around around the copper site.

The plastocyanin class

Plastocyanin is the soluble protein of cyanobacteria (cyanobacteria), algae and plant, and its each molecule contains 1 molecule copper, and its oxidised form is blue.They are present in the chloroplast, and wherein they act as electron carrier.After the structure of determining Cortex Populi dividianae plastocyanin class in 1978, determined the structure of algae (Scenedesmus, Enteromorpha, Chlamydomonas) and plant (Kidney bean) plastocyanin class with crystallography or NMR method, and the structure of Cortex Populi dividianae has been fine to 1.33 Resolution.SEQ ID NO:3 has shown the aminoacid sequence of Phormidium laminosum (a kind of thermophilic cyanobacteria).

Although the sequence divergence between algae and the vascular plant (for example the sequence homogeny between Chlamydomonas and the Cortex Populi dividianae plant is 62%), three dimensional structure be guard (for example, the deviation in the C α site between Chlamydomonas and the Cortex Populi dividianae plant is 0.76

).Architectural feature comprises the tetrahedron copper binding site of the distortion on the end of the antiparallel β bucket of 8 chains, obviously negativity sticking patch and smooth hydrophobic surface.Optimized the electron transfer function in copper site, negativity and hydrophobicity sticking patch have been considered to participate in physiological reaction companion's identification.Chemical modification, crosslinked and direct mutagenesis have been tested empirical tests negativity and hydrophobicity sticking patch with importance during combining of cytochrome f reacts to each other, and confirmed to relate to the model of two significant electron transfer of function of plastocyanin class.Electron transfer of inferring is short relatively by (about 4

) and relate to the copper part His-87 that the solvent on the hydrophobicity sticking patch exposes, then longer (the about 12-15 of another approach

), and relate to nearly conserved residues Tyr-83 (Redinbo et al, J.Bioenerg.Biomembr.26:49-66 (1994)) in the negativity sticking patch.

The Rusticyanin class

Rusticyanin is the single chain polypeptide that contains blue copper that obtains from Thiobacillus (being called sour sulfur bacillus (Acidithiobacillus) now).Determined x-ray crystal structure with the unusual diffraction of multi-wavelength, and it has been fine to 1.9 from the very stable and oxidised form highly oxidized cupredoxin Rusticyanin (SEQ ID NO:4) of thiobacillus ferrooxidant

Resolution.Rusticyanin is made up of core β sandwich folding (the β sheet by 6 chains and 7 chains is formed).Similar to other cupredoxins, that copper ion and cluster are arranged by the distortion tetrahedron four conserved residues (His85, Cys138, His143, Met148) coordination (Walter et al, J.MoI.Biol.263:730-51 (1996)).

False AZURIN. class

False AZURIN. is the single chain polypeptide family that contains blue copper.From the aminoacid sequence of the false AZURIN. of Achromobactercycloclastes shown in SEQ ID NO:5.The x ray structure analysis of false AZURIN. is found that it has the structure similar to AZURIN., although have only low sequence homology between these albumen.Exist two main differences between the population structure of false AZURIN. and AZURIN..A carboxyl terminal extension that comprises two α spirals that is false AZURIN. with respect to AZURIN..At the peptide zone line, AZURIN. contains extended loop, and false AZURIN. shortens to some extent, and it has formed the lobe (flap) that contains short α spiral.Unique main difference in copper atom site is the configuration and the Met-S copper key length of MET side chain, and it is obviously shorter than false AZURIN. in AZURIN..

The Phytocyanin class

The albumen that is accredited as Phytocyanin include but not limited in Fructus Cucumidis sativi basic protein, stellacyanin, Mavicyanin, Umecyanin, peel of Fructus Cucumidis sativi cupredoxin (cucumber peelingcupredoxin), the pea pods the blue copper protein of inferring and from the blue copper protein of arabidopsis.In all these albumen except Fructus Cucumidis sativi basic protein and pea pods albumen, axis (axial) the methionine part that is positioned at the blue copper protein site under the normal condition is replaced by glutamic acid.

Auracyanin

From thermophilic green photosynthetic bacteria Chloroflexus aurantiacus, isolate three little blue copper proteins that are known as Auracyanin A, Auracyanin B-1 and Auracyanin B-2.Two B forms are glycoproteins, and it has performance much at one each other, but are different from the A form.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis finds that apparent monomer molecule amount is 14 (A), 18 (B-2) and 22 (B-1) kDa.

Determine the aminoacid sequence of Auracyanin A, and demonstrated the polypeptide that Auracyanin A is 139 residues (Van Dreissche et al; .Protein Science 8:947-957 (1999)).His58, Cys123, His128 and Met132 are settled in space by expection, as long as they are metal ligands of the evolution conservative in known little cuprein plastocyanin class and the AZURIN., secondary structure prediction shows that also Auracyanin has the general β barrel structure similar to the plastocyanin class of AZURIN. Pseudomonas aeruginosa and Folium Populi Davidianae.But Auracyanin shows the sequence signature of two kinds of little cuprein sequence set.With the overall similarity of the consensus sequence of AZURIN. and roughly the same, be about 30.5% with the overall similarity of the consensus sequence of plastocyanin.The N-terminal sequence area 1-18 of Auracyanin is rich in glycine and hydroxy-amino-acid especially.See example aminoacid sequence SEQID NO:15 (NCBI albumen database numbering No.AAM12874) from the Auracyanin A chain of Chloroflexus aurantiacus.

The cupredoxin that Auracyanin B molecule has standard folds.After deliberation from crystal structure (Bond et al, the J.MoI.Biol.306:47-67 (2001) of the Auracyanin B of Chloroflexus aurantiacus; Incorporate its full content into the application by reference at this).Except additional N-terminal chain, the structure of molecule and antibacterial cupredoxin AZURIN. is closely similar.The same with other cupredoxins, a Cu part is positioned on the chain 4 of polypeptide, and other 3 parts are on the macro ring between chain 7 and 8.With reference to the aminoacid locus of 3 parts in back geometric position, Cu site has been discussed.The Cu land of the Auracyanin B that N-terminal afterbody by showing the significant sequence homogeny of known tethers in the electron transfer protein relevant with some other films may characterize crystallography is tied to the endochylema side of cell membrane.McManus etc. (JBiol Chem.267:6531-6540 (1992)) have provided the aminoacid sequence of B form.See example aminoacid sequence SEQID NO:16 (NCBI albumen database numbering No.1QHQA) from the B chain of the Auracyanin of Chloroflexus aurantiacus.

Stellacyanin

Stellacyanin is a hypotype of Phytocyanin (the omnipresence family of plant cupredoxin).SEQ ID NO:14 has provided an exemplary sequence of stellacyanin.Also known Umecyanin (a kind of stellacyanin) (Koch et al from Radix Cochleariae officinalis, J.Am.Chem.Soc.127:158-166 (2005)) and the crystal structure of Fructus Cucumidis sativi stellacyanin (Hart el al, Protein Science5:2175-2183 (1996)).Albumen has all similar to other Phytocyanin and folds.Ephrin B2 albumen ectodomain quarternary structure and stellacyanin have significant similarity (Toth et al, Developmental Cell 1:83-92 (2001)).In state-run biotechnology information centre albumen database, can find the example aminoacid sequence SEQ ID NO:14 of the stellacyanin that is numbered No.1JER.

The Fructus Cucumidis sativi basic protein

SEQ ID NO:17 has provided the proteic example aminoacid sequence of Fructus Cucumidis sativi alkalescence.Fructus Cucumidis sativi basic protein (CBP) is a kind of 1 type blue copper protein, and its crystal structure has been fine to 1.8

Resolution.Molecule is similar to other blue copper proteins with Greece lace β barrel structure, and difference is that a side of bucket is open, and is called " β sandwich " or " β-taco " (Guss et al., J.MoI.Biol.262:686-705 (1996)) better.EphrinB2 albumen ectodomain quarternary structure and Fructus Cucumidis sativi basic protein have similarity highly, and (the rms deviation of 50 α carbon is 1.5

) (Toth et al, Developmental Cell 1:83-92 (2001)).

The Cu atom has normal blue copper NNSS ' coordination, and the bond distance is Cu-N (His39)=1.93

, Cu-S (Cys79)=2.16

, Cu-N (His84)=1.95

, Cu-S (Met89)=2.61 As if disulfide bond (Cys52)-S-S-(Cys85) played important function in the stable molecule configuration aspects.Polypeptide is folding to be that blue copper protein subtribe (Phytocyanin) and nonmetal albumen artemisiifolia anaphylactogen Ra3 (itself and CBP have the sequence homogeny of height) are peculiar.The albumen that is accredited as Phytocyanin now be in the CBP, stellacyanin, Mavicyanin, Umecyanin, peel of Fructus Cucumidis sativi cupredoxin, pea pods the blue copper protein of inferring and from the blue copper protein of arabidopsis.In all albumen except CBP and pea pods albumen, the axis methionine part that is normally at the blue copper protein site is replaced by glutamic acid.In NCBI albumen database numbering No.2CBP, can find the proteic exemplary sequence SEQ ID NO:17 of Fructus Cucumidis sativi alkalescence.

Using method

The invention provides a kind of the treatment and infected virus or antibacterial and the particularly patient's of HIV-1 infection method, comprise to the patient and use at least a polypeptide that described polypeptide is aforesaid cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents.Expect that also this method can effectively treat the patient who has infected other viruses or antibacterial, such as but not limited to, herpes simplex virus (HSV), Ebola virus, cytomegalovirus (CMV), A, B and C type parainfluenza virus etc., A, B, C, with the G Hepatitis virus, hepatitis D virus (HDV), mumps virus, Measles virus, respiratory syncytial virus, Bunyavirus, arenavirus, the influenza virus sample insect viruses (the orthomyxo-like insect viruscalled Dhori) that are called Dhori, poliovirus, rubella virus, dengue virus; SIV and mycobacterium tuberculosis.

Also known cupredoxin and cytochrome C can be effective to malaria, as described in the application's co-applications.In addition, simultaneously a lot of in the world areas of infected by HIV and malaria are very general, and Africa on the south the Sahara particularly.In some embodiments, the patient who suffers from the HIV infection also suffers from the plasmodium infection.In some embodiments, Therapeutic Method of the present invention also comprises and uses antimalarial drug.In some embodiments, use anti-malaria medicaments jointly.

Method of the present invention comprises mammalian cell is contacted with the compositions that comprises cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents.In some embodiments, mammalian cell has infected virus or antibacterial, for example HIV-1.In other embodiments, mammalian cell will be exposed to virus or antibacterial, for example HIV-1.

The compositions that comprises cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents can be by being exposed to known number of ways and multiple scheme is applied to the patient.In concrete embodiment, by vein, muscle or subcutaneous administration cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents.

In one embodiment, described method can comprise the compositions of using the variant, derivant or the structural equivalents that comprise cupredoxin or cytochrome C 551 or cupredoxin or cytochrome C 551 of a unit dose to the patient altogether, with the compositions of comprising of a unit dose another kind of antiviral or antibacterium and particularly inverase and/or anti-malaria medicaments, order is not limit; Use approximately at the same time, perhaps use the back and use in one preset time, for example use in about 1 minute to 60 minutes after another kind is used, perhaps use in about 1 hour to 12 hours behind another kind of medicament administration at another kind.

In addition, the present invention includes the method that is used to study virus and bacterial infection.In some embodiments, this method comprises that the mammalian cell that will infect virus and antibacterial and cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents contact, and the growth of mensuration infection.In other embodiments, this method comprises mammalian cell and cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents is contacted, described mammalian cell is contacted with virus or antibacterial, and measure the growth of intracellular infection.In some embodiments, antibacterial or virus are HIV, herpes simplex virus (HSV), Ebola virus, cytomegalovirus (CMV), A, B and C type parainfluenza virus, A, B, C and G Hepatitis virus, hepatitis D virus (HDV), mumps virus, Measles virus, respiratory syncytial virus, Bunyavirus, arenavirus, are called influenza virus sample insect viruses, poliovirus, rubella virus, the dengue virus of Dhori; SIV and mycobacterium tuberculosis.In a concrete embodiment, virus is HIV-1, and Bal, 2167 or the RW/92/008/RE1 strain particularly.In other embodiments, mammalian cell is the people.In other embodiments, cell is blood lymphocyte or blood mononuclear cell on every side.In different embodiments, cell is (in vivo) or come in contact at external (in vitro) in animal body.In some embodiments, cell contacts external, then it is imported in the animal body.

The pharmaceutical composition that comprises cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents

For example can produce the pharmaceutical composition that comprises cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents with arbitrary common method by mixing, dissolving, granulating, system ingot, emulsifying, capsulation, embedding or lyophilized method commonly used.Basically the cupredoxin of purification or cytochrome C 551 or its variant, derivant or structural equivalents can easily be combined with pharmaceutically suitable carrier well known in the art.These carriers make goods can be formulated into tablet, pill, lozenge, capsule, liquid, gel, syrup, unguentum, suspension etc.Proper supplementary material also can comprise for example filler and cellulosics.Other dressing for example can comprise aromatic, with toner, antitack agent (detackifiers), thickening agent and other available additives, adjuvant or bonding agent.In some embodiments, the essentially no antiseptic of medicament preparation.In other embodiments, medicament preparation can contain at least a antiseptic.At Ansel et al, Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams ﹠amp; Wilkins, Baltimore MD (1999)) can find universal method in about pharmaceutical dosage forms.

With multiple mode comprise by injection (for example Intradermal, subcutaneous, intramuscular, intraperitoneal etc.), by suck, by local application, by suppository, by using transdermal patch or can using the used compositions that comprises cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents of the present invention by oral.In Ansel et al (the same), can find general information about drug delivery system.In some embodiments, the compositions that comprises cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents can also directly be used the injection that acts on subcutaneous and intravenous injection etc. by preparation.The injection preparation especially preferentially is used for the treatment of patient in the risk that is in virus or bacterial infection, may suffers from or suffer from described infection, the patient that infects of HIV particularly.After for example polypropylene glycol or similar coating agent mix with protective agent, also can oral absorption comprise the compositions of cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents.

When using by injection, cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents can be formulated in aqueous solution, are formulated in physiology's compatible buffers for example Hanks solution, Ringer's solution or normal saline buffer solution specifically.Solution can comprise that preparation medicine for example suspends, stable and/or dispersive medicine.Perhaps, cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents before use can be by for example aseptic constructed powder types of no pyrogen water of suitable carrier.In some embodiments, pharmaceutical composition does not comprise that adjuvant or any other are added into the material that is used to strengthen the immunne response that described peptide stimulates.In some embodiments, pharmaceutical composition comprises the material of inhibition at the immunne response of described peptide.

When using by intravenous fluid, the intravenous fluid that is used to use cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents can be made up of crystal or colloid.At this used crystal aqueous solution that is inorganic salt or other water soluble molecules.Comprise bigger soluble molecule example gel at this used colloid.Intravenous fluid can be aseptic.

The crystalloid fluid that intravenous uses be can be used to and normal saline (0.9% sodium chloride solution), woods lattice lactate buffer solution or Ringer's solution and 5% D/W (being also referred to as D5W sometimes) included but not limited to, as shown in table 2.

Table 2: the composition of crystalloid fluid commonly used

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

When using by suction, for example dichlorodifluoromethane, Arcton 11, carbon dioxide or other suitable gas can discharge cupredoxin or cytochrome C 551 or its variant, derivant or the structural equivalents of aerosol form from the packing of pressurization or aerosol apparatus to utilize suitable propellant.For pressurised aerosol,, the amount that discharges metering can determine dosage unit by being provided.Can make the capsule and the cartridge case of the gel of the mixture of powders that contains albumen and suitable powder substrate such as lactose or starch that is used for inhaler or insufflator.

When using, cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents can be mixed with solution, gel, ointment, emulsifiable paste, gel, suspension etc., as known in the art by local application.In some embodiments, using is mode through transdermal patch.When using is by suppository (for example internal rectum or intravaginal) when being undertaken, and cupredoxin and/or cytochrome C or its variant, derivant or structural equivalents also can be formulated in the compositions that contains suppository base commonly used.

When using when being Orally administered, can easily make cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents by mixing cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents and pharmaceutically suitable carrier well known in the art.Can adopt solid carrier for example mannitol, lactose, magnesium stearate etc.; Suitable carriers makes cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents to be formulated into to be used for tablet, pill, lozenge, capsule, liquid, gel, syrup, unguentum, suspension of the oral absorption of object of being treated etc.For oral solid preparation for example powder, capsule and tablet, suitable excipient comprises filler for example sugar, cellulosics, granulating reagent and bonding agent.

Other common carrier well known in the art also comprise the valency carrier carrier of bacterial capsule polysaccharide, glucosan or hereditism's processing for example.In addition, the extended release preparation that comprises cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents is allowed in the time period that prolongs and is discharged cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents, make that when not having extended release preparation cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents can be removed by objective system and/or be degraded by for example protease and simple hydrolysis before not causing or strengthen the therapeutic effect.

Can prolong or optimize blood flow half-life of compositions of the present invention with certain methods well known in the art, described method include but not limited to the cyclisation peptide (Monk et al, 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)), contain rare amino acid whose peptide (Lee etal, J.Pept.Res.63 (2): 69-84 (2004)), the modification of N and C-terminal (Labrie et al, Clin.Invest.Med.13 (5): 275-8, and Hydrocarbon lock ring (Schafineister et al, J.Am.Chem.Soc.122:5891-5892 (2000) (1990)); Walenski etal, Science 305:1466-1470 (2004)).Xiang Guan method is d-isomerization (replacement) and the stabilized peptide sex modification through D-replacement or L-aminoacid replacement especially.

In various embodiments, pharmaceutical composition comprises carrier and adjuvant (including but not limited to buffer agent, carbohydrate, mannitol, albumen, polypeptide or aminoacid for example glycine, antioxidant, bacteriostatic agent, chelating agen, suspending agent, thickening agent and/or antiseptic), water, oil, saline solution, D/W and glycerite, is similar to other required pharmaceutically acceptable auxiliary substances of physiological status for example buffer agent, toxicity regulator, wetting agent etc.Recognize that though can adopt the known arbitrary suitable carriers of those skilled in the art to use compositions of the present invention, the type of carrier will be according to mode of administration and be different.Utilize technique known also chemical compound can be wrapped in the liposome.Biodegradable microsphere also can be used the carrier of making pharmaceutical composition of the present invention.For example in U.S. Patent No. 4,897, suitable biodegradable microsphere has been described in 268,5,075,109,5,928,647,5,811,128,5,820,883,5,853,763,5,814,344 and 5,942,252.

Pharmaceutical composition can be by known sterilization technology sterilization commonly used or by sterilising filtration.Formed aqueous solution can packaged use or by lyophilized, lyophilized goods can make up with sterile solution before using.

Using of cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents

Can use the cupredoxin or cytochrome C 551 or its variant, derivant or the structural equivalents that are formulated into pharmaceutical composition, and can use with any suitable way, for example by oral, buccal mucosa, suction, Sublingual, rectum, vagina, per urethra, nose, part, percutaneous, promptly percutaneous or intestinal outer (comprising in intravenous, intramuscular, the subcutaneous and coronary artery) are used.Can use the pharmaceutical preparation that can effectively realize any amount of its predeterminated target.More specifically, the compositions of administering therapeutic effective dose.In concrete embodiment, the treatment effective dose generally is from about 0.01 to 20mg/ day/kg body weight.

The chemical compound that comprises cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents can individually or be united other active medicines and is used for the treatment of/prevent HIV and infect or other viruses or bacterial infection.Proper dosage certainly will be according to the character of the cupredoxin that is for example adopted or cytochrome C 551 or its variant, derivant or structural equivalents, host, mode of administration and the pathological changes of being treated and severity different and different.But in general, the dosage from about 0.01 to 20mg/kg body weight will obtain satisfied result human body every day.Given dosage range every day is to use from about 0.7mg to about 1400mg cupredoxin or the chemical compound of cytochrome C 551 or its variant, derivant or structural equivalents in the human body, for example is every day dosage, dosage, every month dosage and/or successive doses weekly.Every day, dosage can be from 1 time to 12 times separate dose every day.Perhaps, can the next day, per 3 days, per 4 days, per 5 days, per 6 days, weekly and similarly natural law is increased to 31 days at interval or longlyer comes application dosage.Perhaps, dosage can be to use the successive doses that paster, intravenous are used etc.

In some embodiments, method from structural equivalents to the patient that introduce cupredoxin or cytochrome C 551 and/or its variant, derivant or is consistent with being used to use the HAART that inverase for example contains protease inhibitor now.These methods are well known in the art.In a concrete embodiment, cupredoxin or cytochrome C 551 and/or its variant, derivant or structural equivalents are the cocktail or the parts of dosage altogether of the other drug of anti-HIV therapy.Other inverase includes but not limited to, reverse transcriptase inhibitors: AZT (zidovudine [Retrovir]), ddC (zalcitabine [Hivid], didanosine), d4T (stavudine [Zerit]) and 3TC (lamivudine [Epivir]), non-nucleoside reverse transcriptase inhibitor (NNRTIS): Delavirdine (Rescriptor) and nevirapine (Viramune), protease inhibitor: ritonavir (Norvir), Lopinavir and ritonavir combination (Kaletra), Saquinavir (Invirase), indinavir sulfate (Crixivan), amprenavir (Agenerase) and viracept see nelfinaivr (Viracept).What at present, be used for the treatment of the patient that carries HIV is a kind of combination that is called some medicines of high activity antiretroviral therapy (HAART).

In some embodiments, method from structural equivalents to the patient that introduce cupredoxin or cytochrome and/or its variant, derivant or is by using antimalarial medicine altogether.These methods are well known in the art.In a concrete embodiment, cupredoxin and/or cytochrome C are parts that contains the cocktail of malaria therapeutic agent or be total to dosage.The therapeutic agent of malaria includes but not limited to proguanil, chlorproguanil, trimethoprim, chloroquine, mefloquine, lumefantrine, atovaquone, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, quinidine, amodiaquine, amopyroquine, sulphonamides, artemisinin, artefiene, artemether, artesunate, primaquine, pyronaridine, proguanil, chloroquine, mefloquine, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, proguanil, chloroquine, mefloquine, 1,16-hexadecamethylenebis (N-methylpyrrolidinium) dibromide, and combination.

Decide correct preparation, route of administration and dosage by the attending doctor according to patient's pathological changes.The amount and the interval of dosage be can adjust to individuation, the active cupredoxin of therapeutical effect or the blood plasma level of cytochrome C 551 or its variant, derivant or structural equivalents are enough to keep to provide.General all using with the mixture of putting into practice selected pharmaceutical carrier through predetermined route of administration and standard drug used required cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents.

In one aspect, cupredoxin or cytochrome C 551 or its variant, derivant or the structural equivalents of transhipment dna form make to generate polypeptide in position.In one embodiment, DNA is " naked " DNA, as described in (Science 259:1745-1749 (1993)) such as Ulmer and as Cohen (Science 259:1691-1692 (1993)) summarizes.By the DNA coating being increased the picked-up to naked DNA to the carrier biological example degradable pearl, naked DNA is by in the transporte to cells effectively then.In these methods, can present DNA with the arbitrary system in the known multiple movement system of those skilled in the art, comprise expression of nucleic acid system, antibacterial and virus expression systems.The technology that is used for DNA is incorporated into expression system is that those skilled in the art are known.See for example WO90/11092, WO93/24640, WO 93/17706 and U.S. Patent No. 5,736,524.

Be used for hereditary material shuttled back and forth from organism and can be divided into two big classes to the carrier of organism: cloning vehicle is reproducible plasmid or phage, and it has duplicate zone necessary and that wherein be inserted into foreign DNA in proper host cell; Foreign DNA is reproducible and breeding, as long as it is an assembly of carrier.Expression vector (for example plasmid, yeast or animal virus genome) is used to exogenous genetic material is incorporated in host cell or the tissue, so that transcribe and translate for example DNA of cupredoxin of foreign DNA.In expression vector, the DNA that is introduced and element for example promoter operably are connected, and promoter is transmitted signal to host cell, so that transcribe the DNA that is inserted high-levelly.Some promoteres are useful especially, inducible promoter for example, and its control responds to the genetic transcription of special factor.The polynucleotide of cupredoxin and its variant, derivant or structural equivalents can be controlled the expression that cupredoxin and/or cytochrome c and its variant, derivant or structural equivalents respond to special factor with operably being connected of inducible promoter.The example of inducible promoter commonly used is those promoteres that respond to interferon-alpha, heat shock, heavy metal ion and steroid such as glucocorticoid (Kaufman, Methods Enzymol.185:487-511 (1990)) and tetracycline.Other required inducible promoters comprise the endogenous promoter of those acellulars, wherein introduce construct in cell, and when derivant was provided, the inducible promoter in these cells was replied when exogenous.Useful expression vector generally usually is a plasmid.But, also pay close attention to for example viral vector (as replication defect type retrovirus, adenovirus and adeno associated virus) of other forms of expression vector.

By used organism or cell and required carrier character decision carrier selection.Carrier generally comprises signal sequence, origin of replication, marker gene, multi-link thing site, enhancer element, promoter and transcription terminator.

The test kit that comprises cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents

In one aspect, the invention provides the test kit that contains the one or more following material in packing or the container: (1) comprises the biological active composite of at least a cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents; (2) antiviral or anti-bacterial drug, inverase particularly, include but not limited to, reverse transcriptase inhibitors: AZT (zidovudine [Retrovir]), ddC (zalcitabine [Hivid], didanosine), d4T (stavudine [Zerit]) and 3TC (lamivudine [Epivir]), non-nucleoside reverse transcriptase inhibitor (NNRTIS): Delavirdine (Rescriptor) and nevirapine (Viramune), protease inhibitor: ritonavir (Norvir), the combination of Lopinavir and ritonavir (Kaletra), Saquinavir (Invirase), indinavir sulfate (Crixivan), amprenavir (Agenerase) and viracept see nelfinaivr (Viracept); (3) pharmaceutically acceptable auxiliaries; (4) mean for applying syringe for example; (5) use description.The embodiment of two or more components (1)-(5) in same package or container is also expected.

In some embodiments, test kit also comprises the malaria therapeutic agent.Interested malaria therapeutic agent includes but not limited to, proguanil, chlorproguanil, trimethoprim, chloroquine, mefloquine, lumefantrine, atovaquone, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, quinidine, amodiaquine, amopyroquine, sulphonamides, artemisinin, arteflene, artemether, artesunate, primaquine, pyronaridine, proguanil, chloroquine, mefloquine, pyrimethamine-sulfadoxine, pyrimethamine-dapsone, halofantrine, quinine, proguanil, chloroquine, mefloquine, 1,16-hexadecamethylenebis (N-methylpyrrolidinium) dibromide.

When test kit was provided, the different component of compositions may be packaged in the isolating container, before use at once with its mixing.This separately packing composition can be allowed long-time storage, and does not lose the function of active component.

Can provide test kit contained reagent with the container of arbitrary type, make the life-span to preserve different component, and do not absorbed or change by the material of container.For example, the sealed glass ampoule can comprise lyophilized cupredoxin and/or cytochrome C or its variant and derivant, the perhaps buffer of having packed under neutral, unresponsive gas such as nitrogen.Ampoule can be composed of any suitable material, for example for example PC, polystyrene etc., pottery, metal or arbitrary material that is normally used for preserving similar reagent of glass, organic polymer.Other examples of suitable vessel comprise conventional bottle, and it can be made by the material similar to ampoule, and shell, and it can comprise the internal layer for example aluminium foil or the Alloy Foil of pressing from both sides paper tinsel.Other containers comprise test tube, bottle, flask, bottle, syringe etc.Container can have the aseptic hole of advancing, and for example has the bottle of the stopper that can be penetrated by hypodermic needle.Other containers can have by separated two chambers of the film of easy taking-up, allow that component mixes after taking out film.The film that can take out can be glass, plastics, rubber etc.

The test kit material that also can furnish an explanation.Description can be printed on paper or other substrates, but the medium of electronic reading such as floppy disk, CD-ROM, DVD-RPM, zip dish, video-tape, audiotape, flash memory disk etc. perhaps can be provided.Detailed description can physically not link to each other with test kit; The substitute is, the user may be directed to the business men of test kit or distributor's specified web or provides by Email.

The modification of cupredoxin and/or cytochrome C and variant thereof and derivant

Cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents can be by chemical modification or hereditism's changes, to generate variant and derivant as explained above.Can synthesize these variants and derivant with standard technique.

Except the naturally occurring allele variant of cupredoxin and cytochrome C 551, can cause change by in cupredoxin or cytochrome C 551 coded sequences, introducing sudden change, this can make the aminoacid sequence of coded cupredoxin change, but can not change virus or bacterial infection and the particularly ability of the growth of HIV infection in cupredoxin or the cytochrome C inhibition mammalian cell significantly." nonessential " amino acid residue is the residue that can be changed and not change biologic activity in the wild-type sequence of cupredoxin, and " essential " amino acid residue is the necessary amino acid residue of this biologic activity.For example, expectation for the amino acid residue of conserved amino acid is not suitable for changing especially, is " essential " aminoacid therefore in cupredoxin.

It is well known in the art can preparing the aminoacid that can not change the conservative replacement of polypeptide active.Table 3 " the preferred replacement " has shown useful conservative replacement.Wherein another aminoacid of same type is within the scope of the present invention to one type amino acid whose conservative replacement, if replace in fact not can the modification compound biologic activity.

Table 3: the preferred replacement

Structure example such as the β sheet or the alpha helical conformation of influence (1) polypeptide backbone; (2) electric charge; (3) hydrophobicity; Or the non-conservative replacement of the volume of (4) the target site side chain function that can both modify cytotoxic factor.Residue can be divided into groups according to the common side chain performance shown in the table 4.The feasible member who exchanges another kind of type with the member in one of these types of non-conservative replacement.Replace and be directed into conservative replacement site or be introduced in non-conservative site more specifically.

Table 4: aminoacid type

Utilize methods known in the art for example oligonucleotide mediated (fixed point) mutation, alanine scanning and PCR mutation can prepare variant polypeptide.(Carter, Biochem be (1986) J.237:1-7 can to implement direct mutagenesis on the DNA that is cloned; Zoller and Smith, Methods Enzymol.154:329-350 (1987)), restricted selection mutation (Wells et al, Gene 34:315-323 (1985)) or other known technology, to generate cupredoxin or cytochrome C 551 modification D NA.

Also can generate variant cupredoxin and the cytochrome C 551 that is used for method of the present invention with known cupredoxin and cytochrome C 551 sudden changes.For example, the C112D of known Pseudomonas aeruginosa AZURIN. and M44KM64E mutant have cytotoxic activity and the growth retardation activity that is different from natural AZURIN., and this activity that has changed can be used for Therapeutic Method of the present invention.An embodiment of the invention are used is the variant and the derivant of the ability of the growth infected of the virus that suppresses in the mammalian cell of cupredoxin and/or cytochrome C 551 and having kept or bacterial infection and particularly HIV.In another embodiment, method of the present invention has been utilized cupredoxin variant such as M44KM64E mutant, and it has the ability that causes the cell growth retardation.

By obtaining with reference to following specific embodiments to understanding more fully of the present invention.Just illustrative purposes for example of embodiment is described, the scope that is not intended to limit the present invention.Provide prompting or the variation of the present invention being made under the situation of purpose for convenience and be equal to replacement and also belong to scope of the present invention.Although adopted concrete term at this, these terms are describing significance, are not the purpose of restriction.Do not breaking away under the spirit and scope of the present invention prerequisite, can carry out as described modification of the present invention and variation before this, therefore scope of the present invention is the scope shown in the appended claims.

Embodiment

Embodiment 1: AZURIN. mutant and cytochrome C 551 at vitro inhibition HIV to lymphocytic infection

The M44KM64E mutant of AZURIN. and cytochrome C 551 to mix (1 micromole/L AZURIN.: 1 micromole/L cytochrome C 551) at 1: 1.(protein concentration is 0,500 to 1000 micrograms/ml) hatched 7 days for people's blood lymphocyte that HIV is infected and blended AZURIN ./cytochrome C 551 albumen.Measure the HIV p24 level in the lymphocyte that infects then.The p24 level is linear relevant with HIV virus levels in the tainted blood.P24 concentration in the mensuration blood can reflect the change of HIV virus titer in the blood.With non-the infected's blood lymphocytes as parallel control.After hatching 7 days, compare with the matched group infection lymphocyte that 0 microgram/ml AZURIN. and cytochrome C 551 are handled, HIV p24 level has descended 25% to 90% in the infection lymphocyte of processed group.At non-infection cellular control unit, hatch 7 days with protein mixture after, do not observe cell death or cellularity occurs.

Embodiment 2. AZURIN. with show structural similarity from the ICAM-I of HIV-1

(the Gough ﹠amp of research in the past; Chothia, Structure 12:917-925 (2004); Stevens et al, J.MoI.Recognit.18:150-157 (2005)) find that cupredoxin and immunoglobulin superfamily member's variable domains has structural similarity.Use DALI algorithm (Holm ﹠amp; Park, Bioinformatics 16:566-567 (2000)) in the 3D data base search from the analog of the AZURIN. (IJZG) of Pseudomonas aeruginosa.See Table 5.AZURIN. not only and with the fab fragment of the coordinate monoclonal antibody of PfMSP1-19 has structural similarity, and have structural similarity (Smith et al., Proc.Natl.Acad.Sci.USA 97:1766-1771 (2000) with the ICAM-I (table 5) that participates in encephalic malaria; Franke-Fayard et al, Proc.Natl.Acad.Sci.USA 102:11468-11473 (2005)).

ICAM-I not only is considered to the receptor of the brain and the capillary endothelium of the tissue of other isolation Plasmodium falciparums (erythrocyte of infection), and see by host cell and carry out HIV-1 granule in the transmittance process, and known its conveying by the enhanced virus material strengthens infectivity (Fortin et al, the J.Virol.71:3588-3596 (1997) of HIV-1; Tardif ﹠amp; Tremblay, J.Virol.77:12299-12309 (2003)).Known ICAM-I can be used as the main group's of rhinovirus and Coxsackie virus receptor (Bella ﹠amp; Rossmann, J.Struct.Biol.128:69-74 (1999)).

AZURIN. and CD4 have structural similarity (table 5), and CD4 is the main host cell surface receptor (Maddon et al., Cell 47:333-348 (1986)) of HIV-1.The present embodiment explanation comprises that the cupredoxin of AZURIN. shows structural similarity because of having 2 antiparallel βZhe Dies that compress face-to-face and connected by disulfide bond with immunoglobulin superfamily member's the variable domains and the extracellular domain and the part thereof of ICAIU (ICAM).

Table 5. Pseudomonas aeruginosa AZURIN. and the proteinic structural similarity of various pathogen dependencys

Use DALI (Holm ﹠amp; Park, Bioinformatics 16:566-567 (2000)) carries out the structure comparison of AZURIN..DALI z integration＜2 that structure is right are considered to dissimilar.

RMSD: the Root-mean-squaredeviation of the framework residue (is unit with the dust) between the right comparison of the structure part.

Embodiment 3. AZURIN., H.8-AZURIN. and Laz be to the influence with viral growth of entering of HIV-1

The AZURIN. of variable concentrations, H.8-AZURIN. and Laz be to the HIV-1 (Bal, RW/92/008/RE1 clade A and IN/2157D15 clade C) of the three kinds of hypotypes influence of growth in the blood mononuclear cell (PBMC) around

The clone of paz and laz gene and expression

Based on the laz gene of its known array (SEQ ID NO:19) clone from Diplococcus gonorrhoeae.Pseudomonas aeruginosa AZURIN. gene (SEQ ID NO:1), be called paz, and from the sequence (SEQ ID NO:18) of the H.8 epi-position of the laz of Diplococcus gonorrhoeae, be used in 5 of paz '-end frame, clone H.8 epitope gene to produce K.8-paz, perhaps in 3 of paz '-end frame, clone H.8 epitope gene with generation paz-K.8.Be used for present embodiment and other bacterial strain and gene constructs about embodiment see Table 6.

Table 6. bacterial strain and gene construct

^*Ap, ampicillin; Km, kanamycin; GST, glutathione S-transferase.

The clone of AZURIN. gene and mistake are expressed existing (Yamada, et al., the Proc.Natl.Acad.Sci.USA 99:14098-14103 (2002) of describing; Punj, et al, Oncogene23:2367-2378 (2004)).The gene (laz) of the Laz of pcr amplification coding Diplococcus gonorrhoeae, the genomic DNA that uses Diplococcus gonorrhoeae bacterial strain F62 is as masterplate DNA.Forward primer that uses and reverse primer be 5 '-CCG GAATTCCGGCAGGGATGTTGTAAATATCCG-3 ' (SEQ IDNO:20) and 5 '-GG GGTACCGCCGTGGCAGGCATACAGCATTTCAATCGG-3 ' (SEQID NO:21), wherein underscore has marked the restriction site EcoRI and the KpnI of extra introducing.The dna fragmentation of amplification is 1.0kb, after EcoRI and KpnI digestion, inserts the corresponding site (Yanisch-Perron of pUC18 carrier, et al., Gene 33:103-119 (1985)), make the laz gene be placed in the downstream of lac promoter, to produce expression plasmid pUC18-laz (table 6).

With pUC19-paz and pUC18-laz as masterplate, by PCR construction expression Diplococcus gonorrhoeae Laz H.8 with the plasmid of AZURIN. (Paz) fusant of Pseudomonas aeruginosa.For fusant H.8-Paz, be that the masterplate amplification obtains the 3.1kb fragment with pUC18-laz, primer is 5 '-(phosphorylation) GGCAGCAGGGGCTTCGGCAGCATCTGC-3 ' (SEQ IDNO:22) and 5 '-CTGCAG GTCGACTCTAGAGGATCCCG-S ' (SEQ IDNO:23), underscore marks the SalI site.With pUC19-paz for the masterplate pcr amplification obtains the 0.4kb fragment, primer is 5 '-(phosphorylation) GCCGAGTGCTCGGTGGACATCCAGG-3 ' (SEQ ID NO:24) and 5 '-TA CTCGAGTCACTTCAGGGTCAGGGTG-S ' (SEQ ID NO:25), what underscore marked is the XhoI site.The clone from the PCR fragment of the SalI of pUC18-laz digestion and from the PCR fragment of the XhoI digestion of pUC19-paz to produce expression plasmid pUC18-H.8-paz (table 6).

Use E.coli JM109 as the host strain of expressing the AZURIN. and the gene of deriving thereof.Reorganization E.coli strain culturing contains 100 micrograms/ml ampicillin, 0.1mM IPTG and 0.5mM CuSO4 in 2X YT culture medium, in 37 ℃ of cultivations 16 hours, to produce AZURIN..

When the E.coli strain growth that carries these plasmids lysis during, go into the described purification AZURIN. of document (Yamada, et al., Proc.Natl.Acad.Sci.USA99:14098-14103 (2002) in IPTG; Punj, et al, Oncogene 23:2367-2378 (2004); Yamada, et al, Cell.Microbiol.7:1418-1431 (2005)), various AZURIN. derivants are moved as single component on SDS-PAGE, unusual migration appears in the protein (about 17kDa) that but contains H.8, as (Cannon, the Clin.Microbiol.Rev.2:S1-S4 (1989) that have reported before this; Fisette, et al, J.Biol.Chem.278:46252-46260 (2003)).

The HIV-1 inhibition analysis

AZURIN., H.8-AZURIN. and Laz be through 0.45 micron membrane filtration degerming.With Polybrene (5 micrograms/ml) handle around blood mononuclear cell (PBMC) 1 hour, be inoculated in titer plate with 250,000 cells/well then.With the centrifugal 5min of plate 800rpm with collecting cell.Remove supernatant, add and contain protein (concentration is 0.3,0.6,1.2,6.0 and 30 micromoles'/L) culture medium (100 microlitre).With cell culture 1h.With AZT (25 micromoles/L) in contrast.Protein is stayed on the cell, added 100 microlitre viruses (Bal, 2167 or RW/92/008/RE1) and hatch 2h.With plate centrifugal 5 minutes of 800rpm once more, remove protein and virus.Add protein and culture medium that cumulative volume is 100 microlitres once more, hatched 5 days.After 5 days finish, by the HIV/p24 of ELISA test cultures supernatant.

The display density as a result of Fig. 1 is that the AZURIN. of 6.0 micromoles/L is about 90% to the inhibition of the growth of HIV-1 Bal (the topmost clade B that is popular in the U.S. and West Europe), clade B Africa separated strain RW/92/008/RE1 and clade C India separated strain IN/2167 D15.But, H.8-AZURIN. (the N end has the H.8 AZURIN. of epi-position) has very high inhibition activity at the low concentration of 0.3 micromole/L to all three kinds of hypotypes, particularly to African hypotype and India's hypotype (Fig. 1).

Neisseria albumen Laz also carries H.8 epi-position (Gotschlich ﹠amp in the N end parts of its Neisseria AZURIN. homologue; Seiff FEMS Microbiol.Lett.43:253-255 (1987); Kawula et al, MoI.Microbiol.1:179-185 (1987)), it is active that it has a similar inhibition to these three kinds of hypotypes, particularly to African hypotype and India's hypotype (Fig. 1), prove this H.8 epi-position can promote the active enhancing of anti-HIV-1 of AZURIN..For all these proteinic concentration, MTT analyzes (Yamada et al, Proc.Natl.Acad.Sci.USA 99:14098-14103 (2002); Punj et al., Oncogene 23:2367-2378 (2004)) all do not observe the dead effect of host cell (PBMC), illustrate that the inhibition to the HIV-1 growth is not caused by host cell death.

Embodiment 4. combines by surface plasma body resonant vibration research AZURIN. and gp120 and CD4's

Carry out surface plasma resonance laboratory to determine AZURIN. and not only be incorporated into CD4 but also in conjunction with the degree of HIV-1 surface protein, these HIV-1 surface proteins such as gp120 or gp41, known participation HIV-1 enters, and other albumen such as Nef or Gag, participates in intracellular virus and duplicates.

Merge the proteic plasmid construction of GST.Use and proofread and correct the plasmid of archaeal dna polymerase by truncate wt-AZURIN. (azu) derivant of polymerase chain reaction construction expression glutathione S-transferase (GST).For pGST-azu 36-128, the amplification PCR fragment is introduced commercially available GST expression vector pGEX-5X, and (Amersham Biosciences, Piscataway is in BamHl NJ) and the EcoRl site.With pUC19-azu as template, utilize primer 5 '-C GGGATCCCCG GCA ACC TGC CGA AGA ACG TCA TGG GC-3 ' (SEQ ID NO:26) and 5 '-CG GAATTCGCA TCA CTT CAG GGT CAG GG-3 ' (SEQ IDNO:27) amplified fragments wherein indicates the BamHl and the EcoRI site of introducing in addition with underscore respectively.(CA) to carry out the c-terminus of azu gene cumulatively truncate by introducing termination codon for Stratagene, La Jolla to use QuickChange rite-directed mutagenesis test kit.

For pGST-azu 36-89, termination codon is introduced among the Gly90.The plasmid that carries pGST-azu36-128 is as template DNA.Three groups of oligonucleotide as rite-directed mutagenesis are as follows.For pGST-azu 36-89:5 '-CCA AGC TGA TCG GCTCGTGAGAGAAGGACTCGGTGACC-3 ' (SEQ IDNO:28) and 5 '-GGTCAC CGA GTC CTTCTCTCACGAGCC GATCAGCTTGG-3 (SEQ IDNO:29).

For pGST-azu 88-113, (CA) to carry out the c-terminus of azu gene cumulatively truncate by introducing termination codon for Stratagene, La Jolla to use QuickChange rite-directed mutagenesis test kit.For pGST-azu 88-113, termination codon is introduced among the Phel 14.The plasmid that carries pGST-azu 88-128 is as template.For pGST-azu 88-128, the amplification PCR fragment is introduced among the BamHI and EcoRI site of commercially available GST expression vector pGEX-5X (Amersham Biosciences).With pUC19-azu as template, use primer 5 '-C GGGGATCCCCG GCT CGG GCG AGA AGG AC-3 ' (SEQ IDNO:30) and 5 '-CGG GAATTCTCC ACT TCA GGG TCA GGG TG-3 ' (SEQID NO:31) amplified fragments wherein indicates the BamHl and the EcoRI site of introducing in addition with underscore respectively.

The one group of oligonucleotide that is used for rite-directed mutagenesis that is used to prepare pGST-azu 88-113 is as follows: 5 '-GTT CTT CTG CAC CTA GCC GGG CCA CTC CG-3 ' (SEQ IDNO:32) and 5 '-CGG AGT GGC CCG GCT AGG TGC AGA AGA AC-3 ' (SEQ ID NO:33).PGST-azu 88-113 is used for Transformed E .coli XL-I Blue bacterial strain.Use commercial reagents box (Qiagen, Venlo, The Netherlands) to carry out plasmid and extract, the performing PCR of going forward side by side order-checking is inserted and transfection with the evaluation plasmid.

E.coli BL21 (DE3) is as the host strain of expressing gst and fusion derivant thereof.Cultivate E.coli strain X L1-Blue3 hour that has transformed the pGST-azu plasmid down adding on the LB culture medium of ampicillin 37 ℃, on culture medium, carry out IPTG to induce (0.4mM), then 37 ℃ down inoculation 2-4 hour so that the expression maximization.Centrifugal separating cell is resuspended to 25mL of I X PBS buffer.Lysis subsequently comprises that two of cell suspending liquid are handled continuously: ultrasonotomography (20 minutes) memory heat-cold shock in acetone-the dry ice bath (using suitable protease inhibitor) on ice.The supernatant of isolated cell cleavage mixture also passes fresh be full of and through the equilibrated 1mL glutathion-sepharose 4B of PBS (Amersham Biosciences) post.Wash post and use pH subsequently be 10mM glutathion eluting GST-azu product among 8 the 20mM Tris-HCl after, use 10% SDS-PAGETris-Gly gel with Coomassie brilliant blue R reagent dyeing by electrophoresis detection GST-Azu 88-113 purity.Use the Bradford method to measure protein concentration.

Surface plasma resonance (SPR) research.(Uppsala, Biacore X spectrometer Sweden) is estimated external protein protein interaction from Biacore AB International in use.Use available from the Au-CM5 sensor chip of Biacore at HBS-EP running buffer (0.01M HEPES, pH 7.4,0.15M NaCl, 3mM EDTA, 0.005% v/v surfactant P20)) in carry out all experiments under 25 ℃.After desalination and lyophilization on the G-75 post, in PBS, prepare the protein stock solution, with pre-concentration and exchange buffering liquid.

Carry out protein immobilization on the CM5 chip according to the amine coupling method.Because the difference of protein cross efficient, after the pre-activation of CM5 surface NHS/EDC, immobilized protein under various conditions: inject 50 μ l AZURIN. (510 μ M), or inject 35 μ l CD4 (25 μ M, 2x), or HIV-1 gp120 (10 μ M).Use ethanolamine (IM, pH 8.8) to handle the CM5 surface then and before in conjunction with research, gone out uncrosslinked protein.When inject finishing, on protein-CM5 surface, carry out combination research by injecting protein eluant (50 μ l) with the flow velocity of 120 seconds time delays, 30 μ l/min.The protein eluant comprises CD4 (Protein Sciences Corp., Meriden, CT), HIV-1 gp120 (Immunodiagnostics Inc., Woburn, MA), HIV-1 gp41 (Bioclone Inc., San Diego, CA), HIV-1 gag and HIV-1-nef (ChemiconInternational, Temecula, CA) and GST-AZURIN. fusion rotein (GST, GST-Azu 36-128, GST-Azu 36-89 and GST-Azu88-113 that the inventor's laboratory is expressed).Between injecting, uses in protein 100mM NaOH (10 μ l injected pulse) regeneration sensor chip surface.All carry out in conjunction with the contrary negative fluid course that contains naked Au-CM5 of research, with the effect of corrigendum non-specific binding.For the combination experiment, wherein CD4 and HIV-1gp120 (not immobilization) play the eluant effect, to the Sensor Chip CM 5 (CarboMer Inc., San Diego CA) of running buffer adding 1mg/mL, to reduce nonspecific proteins matter combination to naked Au-CM5 fluid course surface.

In order to generate the binding constant data, relate to titration experiments by injection and the collection data that increase protein eluant (0.05-2000nM) concentration.The SPR data can be fit to Langmuir balance combination model [Req=Rmax/ (1+Kd/C], determine binding constant (Kd) thus.Similar with aforesaid binding constant research, use similar methods to carry out studying, but inject HIV-1 gp120+ competition albumen (AZURIN., GST-Azu 36-128 and GST-Azu 88-113) with the competition of CD4-CM5.

CD4 is immobilized in the sensor chip, and AZURIN. and gp120 all show remarkable combination the (Fig. 2 A) with CD4.AZURIN. shows affinity in conjunction with CD4 (Kd=36.9nM) and is higher than affinity in conjunction with HIV-1 part gp120 (Kd=48.1nM).Although merging (for example GST-Azu 88-113), the GST-AZURIN. do not show in conjunction with (Fig. 2 A), another GST-AZURIN. fusion rotein GST-Azu 36-128 has shown the combination stronger than AZURIN. self, the Kd value is 0.34nM (a Fig.4A insert), and this shows that the part AZURIN. can keep stronger binding affinity than full length protein.When AZURIN. was immobilized on the sensor chip, gp120 had shown and slightly strong the combining of AZURIN. (Fig. 2 B) that than CD4 this clearly illustrates that AZURIN. is with high-affinity gp120 and CD4.What is interesting is, also participate in gp41 that HIV-1 enters host cell and do not show any combination (Fig.2B) with AZURIN..Similarly lack in conjunction with also showing Gag and Nef.

Embodiment 5. combines by surface plasma resonance research AZURIN. and ICAM and CD5's

AZURIN. and known the infection between the relevant ICAM (table 5) with receptor HIV-1 exist structural similarity (Liao et al. .AIDS Res.Hum.Retroviruses 16:355-366 (2000); Hioe et al, J.Virol.75:1077-1082 (2001)).ICAM-3 is stimulated HIV-1 to transcribe and virus production by hint, therefore helps born of the same parents' inner virus growth (Barat et al, J.Virol.78,6692-6697 (2004)) in addition.

Thereby, studied by the AZURIN. of SPR mensuration and the protein protein interaction between the ICAM (as ICAM-I, ICAM-2, ICAM-3 and NCAM).Utilization is immobilized AZURIN. on the CM5 chip, and (Fig. 2 C, Kd=19.5 ± 5.4nM) and NCAM (Fig. 2 C, illustration) rather than ICAM-I and ICAM-2 have shown strong combination to ICAM-3.Although be not intended to limit the invention to arbitrary mechanism, AZURIN. also can be regulated by the interaction between itself and ICAM-3 or the NCAM the inhibition part of HIV-1 growth.

Embodiment 6. is by surface plasma resonance research AZURIN. and gp120 competition CD4

Because AZURIN. and gp120 compare the high-affinity (Fig. 2 A) of CD4, whether the experiment that is at war with can disturb gp120 and its combining with receptoroid CD4 to observe AZURIN..Under the situation of the concentration fixed that is adsorbed onto the gp120 on the immobilized CD4 chip, along with competition albumen (AZURIN., GST-Azu 36-128 or GST-Azu 88-113) concentration increases, AZURIN. and GST-Azu 36-128 all show from the bonded remarkable reduction of the gp120 of CD4-CM5 chip total protein (Fig. 2 D).For GST-Azu 88-113, do not observe this significantly from chip displacement gp120 (Fig. 2 D).Known GST-Azu 88-113 is not in conjunction with CD4 (Fig. 2 A).Although be not intended to limit the invention to arbitrary mechanism, this shows that AZURIN. or GST-Azu 36-128 fusion rotein can successfully suppress to form complex between gp120 and the CD4.

Embodiment 7. combines by surface plasma resonance research AZURIN. and ICAM-3 and DC-SIGN's

AZURIN. and strong combination the (Fig. 2) of gp120, CD4 and ICAM-3 imitated another that be known as DC-SIGN (DC-specificity intercellular adhesion molecule 3-is in conjunction with the nonconformity element) be present in the lip-deep very important HIV-1 of dendritic cell (DC) conjugated protein with the combining of the associated protein that is called DC-SIGN/R.DC-SIGN expresses on DC galore, and DC-SIGN/R mainly expresses on hole cell and endotheliocyte.The antigen-presenting cell DC of DC-SIGN by making specialty through they with the T cell surface on ICAM-3 between interaction catch and present the antigen static T cell extremely that comprises HIV-1, and (the Geijtenbeek et al that in the HIV-1 immunopathogenesis, plays a major role, Cell 100,575-585 (2000); Soilleux, Clin.Sci.104,437-446 (2003); Geijtenbeek et al, Placenta 22, S19-S23 (2001)).DC-SIGN also shows with HIV-coating protein gp120 and combines actively, catches HIV-1 thus and it is transferred to the CD4+T cell, can freely duplicate (Snyder etal, J.Virol.79:4589-4598 (2005)) at this HIV-1.

In immobilized DC-SIGN carries out on sensor chip the SPR experiment, AZURIN. (Kd=0.83 ± 0.05nM) and ICAM-3 (Kd=0.93 ± 0.39nM) all combine (Fig. 3 A) by force with DC-SIGN.And GST merges the very little combination (Fig. 3 A) of derivant GST-Azu 36-89 demonstration, another GST-merges derivant GST-Azu 88-113 and presents strong relatively combination (Kd=5.98 ± 1.13nM), show that the C-end portion of AZURIN. has participated in DC-SIGN in conjunction with (Fig. 3 B).Yet GST-Azu 88-113 shows that not in conjunction with CD4 (Fig. 2 A) different piece of AZURIN. has different binding specificities.

Although be not intended to limit the invention to arbitrary mechanism, with the bonded potential of clear AZURIN. interference HIV-1 of this associative list of DC-SIGN and DC.Therefore, DC-SIGN is responsible on a kind of DC surface HIV-1 is transferred to the important molecule of lymph sample T cell by mucomembranous cell, can same active in conjunction with gp120, CD4 and ICAM-3 AZURIN. or Laz in find strong competitor well.

Embodiment 8. AZURIN ./Laz worked in the stage that enters that HIV-1 infects

For determine AZURIN. whether entering of infecting of HIV-1 or after enter step and work, studied effect to the India separator IN/2167 of HIV-1.In an experiment, inoculate activatory PBMC (25,000 cells/well) 2 hours with 6.0 micromoles/L Laz and HIV-1.Centrifugal mixture is to remove Laz and HIV-1, and add-back does not have the fresh culture of Laz, and cultivates this culture 5 days.By measuring the p24 monitoring HIV-1 growth in the culture supernatant.Under this condition, (6.0 micromoles/L) suppress 43% of HIV-1 growth to Laz.(30 micromoles/L), the inhibition degree is 76% to utilize the Laz of higher concentration.In parallel laboratory test, after HIV-1 infects and is removed with Laz (6 or 30 micromoles/when L) adding PBMC, observe the inhibition of very little viral growth.As positive control, when Laz (6.0 micromoles/L) be present between infection period and remove virus removal with fresh culture after between 5 days culture periods the time, the inhibition degree is about 93%.Although be not intended to limit the invention to arbitrary mechanism, such data clearly illustrate that AZURIN. or Laz mainly exert an influence in the stage that enters of infecting.

Embodiment 9: with the patient of AZURIN. treatment infected by HIV

Record 24 patients that carry AIDS by round pcr and in blood plasma, present low CD4+ counting undetectable HIV viral load (RNAPCR) and increase.Then, separate and FACS classification enrichment CD4+RO+ cell by magnetic, and measure to determine appeal about natural non-infected cells co-culture experiments.There is infectious HIV in the analysis showed that of this CD4+RO+ memory cell.

Therefore, inject with 4mg/m by intravenous ²Dosage whenever biweekly with 20 3 weeks of patient of AZURIN. administration, know that the CD4+ cell that comprises memory cell is in low-level.4 patients are subjected to placebo and inject.During the administration AZURIN., or get over 1-2 month thereafter, or restore, keep the patient with antibiotic and antifungal therapy up to the CD4+ cell.Provide stem cell or precursor by bone marrow transplantation and cytokine therapy, both are all undertaken by routine techniques.

During the treatment and after the treatment, follow up a case by regular visits to the patient and detect CD34 cellular level, the reconstruction of CD4+ cell and quantification CD4+RO+ cell with frequent interval.In addition, the viral load by co-culture of cells measuring patient blood plasma.Reduce viral load and be reduced to low concentration or can not detectable concentration the time, stop to use AZURIN. activity being remembered the CD4+T cell to the patient.After 3 months, stop to patient's administration of antibiotics and antifongin therapy.After this, follow up a case by regular visits to the patient, and measure viral level with 6 months interval.The result has shown the effectiveness of AZURIN. therapy to the HIV infected patient.

Claims (38)

1. isolating peptide, it is variant, derivant or the structural equivalents of cupredoxin or cytochrome C 551, and can suppress the growth that HIV-1 infects in the mammalian cell.

2. the isolating peptide of claim 1, wherein said cupredoxin is selected from AZURIN., false AZURIN., plastocyanin, rusticyanin, Laz and auracyanin.

3. the isolating peptide of claim 2, wherein said cupredoxin is AZURIN. or Laz.

4. the isolating peptide of claim 1, wherein said cupredoxin are from the organism that is selected from as next group: 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 parahaemolytious (Vibrio parahaemolyticus).

5. the isolating peptide of claim 4, it is from Pseudomonas aeruginosa or Diplococcus gonorrhoeae.

6. the isolating peptide of claim 1, it is a part that is selected from the peptide among the SEQ ID NO:1-17.

7. the isolating peptide of claim 1, being selected from by the sequence of SEQ ID NO:1-17 and described peptide has aminoacid sequence homogeny at least about 90%.

8. the isolating peptide of claim 1, it is the clipped form of cupredoxin or cytochrome C 551.

9. the isolating peptide of claim 1, wherein said peptide have about 10 residues of surpassing but are no more than about 100 residues.

10. the isolating peptide of claim 9, wherein said peptide comprises the AZURIN. district that is selected from residue 36-128, residue 36-88 and residue 88-113.

11. the isolating peptide of claim 10, wherein said peptide is made up of the AZURIN. district that is selected from residue 36-128, residue 36-88 and residue 88-113.

12. the isolating peptide of claim 1, wherein said peptide comprise the residue of cupredoxin with the AZURIN. district equivalence that is selected from residue 36-128, residue 36-88 and residue 88-113.

13. a compositions, it comprises the peptide of at least a cupredoxin, cytochrome C 551 or the claim 1 that are present in the pharmaceutical composition.

14. the compositions of claim 13, wherein pharmaceutical composition is configured to and is used for intravenous and uses.

15. the compositions of claim 13, wherein said cupredoxin is from the organism that is selected from Pseudomonas aeruginosa, alcaligenes faecalis, Achromobacter xylosoxidans, bordetella bronchiseptica, methylomonas, Neisseria meningitidis, Diplococcus gonorrhoeae, pseudomonas fluorescens, Pseudomonas chlororaphis, xyllela fastidiosa and vibrio parahaemolytious.

16. the compositions of claim 15, wherein said cupredoxin is from Pseudomonas aeruginosa or Diplococcus gonorrhoeae.

17. the compositions of claim 13, wherein said cupredoxin or cytochrome C 551 are selected from SEQ ID NO:1-17.

18. the patient's of virus or antibacterial method has been infected in treatment, comprises the compositions to the claim 13 of described patient's administering therapeutic effective dose.

19. the method for claim 18, wherein said patient infection be selected from pathogen with next group: HIV-1, herpes simplex virus (HSV), Ebola virus, cytomegalovirus (CMV), A, B and C type parainfluenza virus, A, B, C and G Hepatitis virus, hepatitis D virus (HDV), mumps virus, Measles virus, respiratory syncytial virus, Bunyavirus, arenavirus, the upright virus of assistant, poliovirus, rubella virus, dengue virus; SIV and mycobacterium tuberculosis.

20. the method for claim 19, wherein said patient infection HIV-1.

21. the method for claim 18, wherein said patient is the people.

22. the method for claim 18 is wherein by being selected from the mode applying said compositions with next group: intravenous injection, intramuscular injection, subcutaneous injection, suction, local application, transdermal patch, suppository and oral.

23. the method for claim 22, wherein method of application is by intravenous injection.

24. the method for claim 18, wherein applying said compositions in using 0 minute to 1 week of at least a medicine that is selected from antiviral drugs, anti-bacterial drug and the inverase.

25. the method for claim 24 is wherein with the about applying said compositions simultaneously of another kind of inverase.

26. a compositions, it comprises at least two kinds of isolating polypeptide that are selected from as in next group: variant, derivant or the structural equivalents of cupredoxin, cytochrome C 551 and cupredoxin or cytochrome C 551.

27. the compositions of claim 26, it is present in the pharmaceutical composition.

28. a test kit, it comprises the compositions that is present in the claim 13 in the bottle.

29. the test kit of claim 28, wherein said test kit designed to be used intravenous and uses.

30. a method comprises cell and cupredoxin or cytochrome C 551 or its variant, derivant or structural equivalents are contacted; Mammalian cell is contacted with antibacterial or virus; Growth with the infection of measuring described virus or antibacterial.

31. the method for claim 30, wherein cell and cupredoxin or cytochrome C 551 or the contacted step of its variant, derivant or structural equivalents betide before cell and antibacterial or the viral contacted step.

32. the method for claim 30, wherein cell and cupredoxin or cytochrome C 551 or the contacted step of its variant, derivant or structural equivalents betide after cell and antibacterial or the viral contacted step.

33. the method for claim 30, wherein cell is the human cell.

34. the method for claim 30, wherein cell is selected from lymphocyte and blood mononuclear cell on every side.

35. the method for claim 30, wherein said virus or antibacterial are selected from: HIV-1, herpes simplex virus (HSV), Ebola virus, cytomegalovirus (CMV), A, B and C type parainfluenza virus, A, B, C and G Hepatitis virus, hepatitis D virus (HDV), mumps virus, Measles virus, respiratory syncytial virus, Bunyavirus, arenavirus, the upright virus of assistant, poliovirus, rubella virus, dengue virus, SIV and mycobacterium tuberculosis.

36. the method for claim 35, wherein said virus is HIV-1.

37. an expression vector, the peptide of its coding claim 1.

38. an isolating peptide, it is variant, derivant or the structural equivalents of cupredoxin; And can be in conjunction with the protein that is selected from CD4, gp120, ICAM3 and DC-SIGN.

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