CN101501210A - System and compositions for isolating suppressors of HIV-1 protease - Google Patents

Abstract

The present invention relates to a yeast screening assay for the identification of suppressors of a protease, such as the HIV-I protease and/or HIV-2 protease, for example. Upon overexpression of HIV-I protease in Schizosaccharomyces pombe, for example, the cell dies, but in the presence of a candidate inhibitor with protease suppressor activity, the cell lives, thereby providing an assay for the identification of one or more suppressors. In additional aspects, there is an in vivo protease activity screen, used alternatively or additionally to a viability screen, such as for cleavage of a HIV-I protease-specific site. In further aspects, the suppressor is employed for the treatment of an individual infected with HIV virus or with AIDS.

Description

The system and the pharmaceutical composition that separate the HIV-1 proteinase inhibitor

It is that June 9, application number in 2006 are the right of priority of 60/812,685 U.S. Provisional Patent Application that the present invention has required the applying date, and the full content of this U.S. Provisional Patent Application is incorporated herein this paper as a reference.

About the statement of the present invention for the scientific research project of federal funding: this invention has obtained the subsidy of NIH (NIH), and project approval number is NIH NIGM R01 GM63080 and N1H-N1AID R01AI40891.United States Government has certain power to the present invention.

Technical field

The present invention relates to cytobiology, molecular biology and medical field at least, particularly the pharmaceutical composition of treatment HIV/AIDS.

Background technology

HIV/AIDS still is the disease that can not cure, threatens global millions of people's life.Drug cocktail therapy (treatment) generally also claims HAART or ART, is to make patient's HIV-1 be reduced to the most successful antiretroviral therapy under can detected level at present.Typical A RT therapy need be united a use HIV-1 proteinase inhibitor (HIV-1PI) and two HIV-1 reverse transcriptase inhibitors.In two class anti-HIV medicines, HIV-1 PI is the most effective viral inhibitors, and its independent use also can make virus reduce by 2-3 the order of magnitude.Hiv protease is produce virus infectivity basic, and it can cut into viral poly precursor protein challenge virus enzyme (reversed transcriptive enzyme, proteolytic enzyme and intergrase) and structural protein.Therefore, HIV-1 proteolytic enzyme is used to produce the structural protein and the enzyme of virus, and also is necessary for the maturation of infectious virus particle.At present, the PI one that obtains drugs approved by FDA has 8, and these all PI belong to and protease activities site bonded competitive inhibitor, and in fact, these many PI wherein are the couplings that is designed to be able to protease activities site structure.PI stops proteolytic enzyme cutting virus precursor polymerization polypeptide with combining of protease activities site, thereby has blocked virus maturation subsequently.One of subject matter that the ART therapy faces is that virus can be fast produces resistance to medicine, the PI resistance of virus generally be since in the proteinase gene progressively the cumulative sudden change occur, and this resistance increases rapid.ART treatment failure often is that multidrug resistance causes, and promptly proteolytic enzyme becomes has resistance to the most or whole PI that use, and this warns our HIV multidrug resistance finally might surpass our obtainable PI quantity at present.Thereby other can have the PI of drug effect to drug-fast proteolytic enzyme is arranged to need exploitation in a hurry at present.

Summary of the invention

The present invention relates generally to a kind of screening of and HIV medicine and uses relevant system, method and pharmaceutical composition.

Particularly, the contriver has obtained certain progress in this area, and the standard method of feasible screening HIV-1 proteinase inhibitor is developed and utilizes, and this method also can replace to the screening method of HIV-2 proteinase inhibitor certainly.In first embodiment that present specification provides, the proteinase gene high level expression of fission yeast Yin Qinei and causing death, this is found to be differentiates that proteinase inhibitor provides the foundation, when proteinase gene is expressed, the ability that makes cell survival growth that can have by proteinase inhibitor and it is separated.

In specific embodiments more of the present invention, the standard screening method based on cell is provided, this method can identify the compound that can suppress wild-type simultaneously and have the proteolytic enzyme of resistance.These new inhibitor in some cases may not can combine with avtive spot, therefore for we have disclosed the new way of arrestin enzymic activity, promptly pass through other zones and the functional zone of targeting proteins enzyme.Particularly, the present invention can adopt some tests based on the fission yeast cell in full-automatic high-throughput molecular screening (HTS) method of the wide spectrum inhibitor that is used for screening HIV-1 proteolytic enzyme.More specifically, these tests can be used 96 orifice plate patterns and compare the pattern of miniaturization.More specifically, the detection of protease activity can adopt light absorption ratio to detect cell density, perhaps also can adopt the fluoroscopic examination cytoactive, as can be with fluoroscopic examination as further confirmatory test.Other or the alternate embodiment in, adopt GFP-substrate-Vpr fusion rotein carried out the reverse shaker test that the green fluorescence reorientation is used as detecting protease activity.More specifically, these three kinds of detection methods can be applied in the HTS pattern and/or utilize some potential inhibitor libraries, for example the LOPAC1280 library of compounds of Sigma.

In some content of the present invention, also provide in the come up method of Screening and Identification proteinase inhibitor of several levels.For example, when proteinase gene is expressed, cytoactive is screened the proteinase inhibitor of differentiating that some can make cell continuation survival growth as screening index.Screening method based on another level, use the method for external enzymatic assays protease activity to screen proteinase inhibitor, this method can be united use with the cytoactive measuring method, also can substitute the cytoactive measuring method, for example the enzymolysis protein enzyme substrates.Though restriction enzyme site can be the appropriate site that any HIV-1 can enzyme cuts in general embodiment, in specific embodiment, the restriction enzyme site sequence is sequence shown in SEQ ID NO:3 or the SEQ ID NO:4 preferably.

In another embodiment, adopt the method for external test protease activity, protease activity is located in cell by green fluorescent protein (GFP) and is measured.This measuring method provides the method for a semiquantitative determination intracellular protease gross activity, and the substrate specificity that can be used in proteolytic enzyme carries out qualitative.

With regard to many embodiments of the present invention, for example, the separation inhibitor can remove the background because of the cell of homologous recombination loss of expression gene on the one hand.For example, will the polynucleotide that microbiotic (as G418) has a resistance be connected with the proteolytic enzyme polynucleotide, these cells that lose proteinase gene can be differentiated at an easy rate and are removed in the proteinase inhibitor screening.The background that these means cause genetically deficient drops to low-down level, to such an extent as to it no longer can obviously disturb the screening of proteinase inhibitor.

The contriver is a standard screening method of the present invention, is about to have the program that low background and successful Application are crossed, and is applied in the method for screening fission yeast genomic library, and having identified the hhp2 gene is the proteinase inhibitor of fission yeast.In the prior art, without any can being reported qualitatively about relation or inhibition mechanism between hhp2 and the HIV-1 proteinase gene.Therefore, the hhp2 polynucleotide is that a new standard suppresses mechanism, and this finds also to provide new strategy for research suppresses HIV-1 proteolytic enzyme.

In one embodiment of the invention, a kind of method of differentiating the hiv protease inhibitor is provided, this method may further comprise the steps: a kind of yeast cell at first is provided, includes the polynucleotide of being made up of the gene order of coding hiv protease in the described yeast cell; Then described polynucleotide is inserted in the candidate agent; Reevaluate following wherein one or two indexs: 1) cytoactive of described yeast cell and/or growing state; Wherein, compare with cell under the situation that lacks candidate agent, cell can be survived under the situation that described candidate agent exists, and described candidate agent is the hiv protease inhibitor; 2) enzyme of hiv protease substrate is cut effect, and wherein can stop the digested candidate agent of substrate is the hiv protease inhibitor.More specifically, hiv protease is HIV-1 proteolytic enzyme or HIV-2 proteolytic enzyme.In another particular of the present invention, the adjusting of expression regulation sequence has been in the expression of hiv protease.Further, these inhibitor can be polypeptide, polynucleotide, small molecules, antibody, perhaps the mixture or the composition of above multiple material.The preferred fission yeast of yeast cell, for example schizosaccharomyces pombe.

In particular of the present invention, polynucleotide is integrated in the yeast genes group.Crossing expression regulation sequence and can select nml1 to cross expression regulation sequence, adh1 regulating and controlling sequence, fbp1 regulating and controlling sequence, inv1 regulating and controlling sequence, perhaps also can be the ctr4 regulating and controlling sequence in some cases.Polynucleotide can further preferably comprise at least one fragment that can reduce the background growth signals in the cytoactive test.In specific embodiment, the component that reduces background can be marker, for example can for resist marker that living element has resistance, to G418 or Totomycin have resistance marker, nutritional marker, also can be selected from ade1, ade6, arg3, CAN1, his3, his7, leu1, leu2, sup3-5, ura3 and ura3.

In some embodiment, hiv protease behaviour hiv protease, hiv protease substrate behaviour hiv protease substrate.More specifically, the enzyme of assessment hiv protease substrate is cut effect can cut effect for the enzyme that assessment is formed polypeptide by first polypeptide fragment and second polypeptide fragment, and wherein the restriction enzyme site of hiv protease is between described first polypeptide fragment and second polypeptide fragment.On the one hand, can be detectable mark in first polypeptide fragment, on the other hand, second polypeptide fragment can be the substrate of hiv protease, for example HIV-1Vpr polypeptide.

Particularly, restriction enzyme site is DSQNYPIVQ (referring to SEQ ID NO:3), DSFNSTQIT (referring to SEQ ID NO:4), VSQNYPIVQN (referring to SEQ ID NO:8), KARVLAEAMS (referring to SEQ ID NO:9), SATIMMQRGN (referring to SEQ ID NO:10), RPGNFLQSRP (referring to SEQ ID NO:11), VSPNFPQITL (referring to SEQ ID NO:12), CTLNFPISPI (referring to SEQ ID NO:13), GAETFYVDG (referring to SEQ ID NO:14) or IRKVLFLDGI (referring to SEQ ID NO:15).Preferably, detectable mark can be green fluorescent protein, emerald green fluorescin, yellow fluorescence protein, blue fluorescent protein or cyan fluorescent protein.Preferably, this method can further include the described inhibitor of will treat effective dose and is transported to the step of carrying HIV or suffering from the individuality of AIDS.In addition preferably, this method comprises further yet that described inhibitor with effective dose is transported to PI HIV virus and maybe may suffers from the step that the individuality of AIDS is used as preventing.

In one embodiment of the invention, provide a kind of treatment to carry HIV or suffered from the individuality of AIDS, this method comprise will the treatment effective dose the pharmaceutical composition that includes hhp2 be transported to the intravital step of patient.Particularly, hhp2 is a kind of polynucleotide, the polynucleotide of optimized encoding hhp2 polypeptide.In the sequence table shown in SEQ ID NO:16 (

Accession No.U10864) provides the standard sequence of the hhp2 polynucleotide of schizosaccharomyces pombe.The standard sequence of schizosaccharomyces pombe hhp2 polypeptide referring to SEQ ID NO:17 in the sequence table (

Accession No.AAA21545) shown in.From protein sequence and structure, fission yeast hhp2 and human casein kinase 1 (CK1) homology; The standard sequence of CK1 polynucleotide referring to sequence table SEQ ID NO:18 (

Accseeion No.X80693), the standard sequence of CK1 polypeptide referring to sequence table SEQ ID NO:19 (

Accession No.CAA56710).Pharmaceutical composition of the present invention, as the medicament that is used for the treatment of HIV or AIDS has HIV-1 and/or HIV-2 proteinase inhibitor function, for example its sequence may be for shown in sequence table SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or the SEQ ID NO:19, perhaps includes the molecule that has at least 70%, 75%, 80%, 85%, 90%, 95% or 100% similarity with sequence shown in sequence table SEQ ID NO:16 or the SEQ ID NO:17 in these pharmaceutical compositions.

In a specific implementations of the present invention, provide a kind of yeast cell that includes the polynucleotide of the hiv protease of encoding.Particularly, preferably HIV-1 proteolytic enzyme or HIV-2 proteolytic enzyme of hiv protease.Particularly, preferably the polynucleotide of the control of expression regulation sequence has been in its expression.Again further, polynucleotide can be marker, for example microbiotic is had the marker of resistance, further preferred nutritional marker.On the other hand, also can further include the polynucleotide of being made up of second sequence of first sequence of coding first polypeptide fragment and second polypeptide fragment of encoding in the yeast cell, wherein the restriction enzyme site of hiv protease is between first polypeptide fragment and second polypeptide fragment.Further, include detectable mark in first polypeptide fragment, for example green fluorescent protein, emerald green fluorescin, yellow fluorescence protein, blue fluorescent protein or cyan fluorescent protein; Second polypeptide fragment is the preferred Vpr polypeptide of HIV-1 also.In addition, yeast cell can be yeast cell culture or yeast cell clone.

In an additional embodiment of the present invention, the pharmaceutical composition test kit that treats and/or prevents infected by HIV and/or suffer from AIDS is provided, include polynucleotide or the hhp2 polypeptide of the coding hhp2 that is contained in the suitable containers in this test kit.Particularly, this test kit can also further comprise pharmaceutically acceptable excipient.In another concrete additional embodiment of the present invention, provide the test kit that is used to screen one or more hiv protease inhibitor, included yeast cell of the present invention in this test kit.More specifically, yeast cell of the present invention can also further include the polynucleotide of being made up of second sequence of first sequence of coding first polypeptide fragment and coding second polypeptide fragment, and wherein the restriction enzyme site of hiv protease is between first polypeptide fragment and second polypeptide fragment.

Foregoing has been listed abundant technical characterictic of the present invention and technical progress, can help to understand the following detailed description of the invention.Other technical characterictic and technical progress of the present invention will be introduced hereinafter, and these also become the claimed theme in claims of the present invention.All equivalences of doing according to the inventive concept that provides in the present patent application file and embodiment change or modify, and all are that those skilled in the art can expect; Same, every invention that is equal to that belongs to spirit of the present invention and the determined scope of claims also is that those skilled in the art can expect, all should be encompassed in protection scope of the present invention.In order to allow those skilled in the art can understand inventive point of the present invention, composition of the present invention and implementation method and further improved technical scheme and technical progress better, the present invention will be described in detail below in conjunction with embodiment and accompanying drawing.Yet embodiment that provides and accompanying drawing content only for explaining and explanation the present invention, can not be considered as limiting the scope of the invention.

Description of drawings

For a better understanding of the present invention, list description of drawings below as a reference.

Fig. 1 shows when screening inhibitor under high background, removes integrase gene by homologous recombination;

Fig. 2 shows near the kalamycin resistance gene the proteolytic enzyme, is used to the disappearance of differentiating that homologous recombination causes.

Fig. 3 shows the plasmid that is integrated into the nmt1 position by homologous recombination;

Fig. 4 shows isolating two strain G418 resistant strains, and their growth is by strongly inhibited when proteolytic enzyme is expressed;

Fig. 5 illustrates and induces the cell that does not form the clone on the flat board;

Fig. 6, proteolytic enzyme lose activity after expressing and being suppressed;

Fig. 7 uses GFP-Vpr fusion rotein reorientation monitoring protease activity;

Fig. 8, HIV-1 proteolytic enzyme does not influence the location of GFP;

Fig. 9, proteolytic enzyme do not make GFP-Vpr be repositioned onto tenuigenin;

Figure 10, proteolytic enzyme make the GFP among the GFP-SL Ma-Vpr navigate to tenuigenin once more;

Figure 11, proteolytic enzyme make the GFP among the GFP-SL p6-Vpr navigate to tenuigenin once more;

Figure 12, proteolytic enzyme do not make the GFP among the GFP-SL A-Vpr navigate to tenuigenin once more;

Figure 13 compares with the PrIntB bacterial strain, and the growth of PrIntB G418 resistant strain is suppressed;

Figure 14, although nmt1 promotor difference, two typical strains all have single plasmid integration to enter the nmt1 site;

Figure 15 reflects protease activity in the mode of GFP per-cent (GFP%);

Figure 16, indinavir (indinavir) proteolytic enzyme is to the effect of growth;

Figure 17, indinavir (indinavir) stop the GFP among the GFP-SL p6-Vpr to re-position at tenuigenin;

Figure 18, along with the increase of indinavir (indinavir) concentration, GFP reduces;

Figure 19, indinavir (indinavir) promote clone's spot to form;

Figure 20 has only 1 can induce formation clone on the flat board in 1000 cells;

Figure 21 induces the great majority clone on the flat board to lack kalamycin resistance;

Figure 22, the clone that the G418 replicon is originated tests again, shows that great majority have proteinase gene;

Figure 23 uses the PrIndA bacterial strain to carry out the low background screening of proteinase inhibitor;

Figure 24, background comprises G418 resistance and the fierce growth on the T flat board for example;

Figure 25, quick growth on the T substratum and plasmid are irrelevant;

Figure 26 induces dull and stereotyped background bacterial strain and the equal proteinase gene of normal strains of going up growth that phase shift mutation is all arranged;

Figure 27, the separation of weak inhibitor plasmid;

Figure 28 depends on the weak inhibition of plasmid;

Figure 29, after intestinal bacteria were brought back to life, a typical plasmid (pPS3-3) is resurveying as other not;

Figure 30,11 10 of bringing back to life in the plasmid detect again, are the weak inhibitor of growth;

Figure 31,11 10 of bringing back to life in the plasmid detect again to increasing the weak inhibitor of cytoactive;

Figure 32,10 typical inhibitor plasmids all contain the hhp2 gene.

Detailed description of the invention

I. definition

In order to be consistent the english vocabulary that uses in specification of the present invention and claims " a " and " an " expression " one or more " with the Patent Law convention that exists for a long time always. May comprise or originally just comprise one or more components, step and/or method in the some embodiments of the present invention. Additive method or composition that any method that provides in the present specification or composition also can the alternative costs those skilled in the art can be expected.

II. implementation explanation of the present invention

HIV/AIDS is one of disease of global most threatening property, and nearly 4,000 ten thousand people in 2005 infect or carry HIV, and 4,000,000 new the infecteds are arranged every year approximately. In the U.S., HIV is 25 to 44 years old adult's second largest killer. HIV-1 protease is the drug targets that clearly the most successful treatment HIV infects. Protease-producing strain is in an inactive Gag-Pol precursor polymeric albumen, and frameshit is read and produced in translation process. Protease discharges the necessary various albumen that comprise complete active protease of infectious virus assembling at 9 place's scinderins of polymeric protein. Since the protease mechanism is the important step that produces virion, protease has become the main target spot of inverase, and protease inhibitors (PI) is the main policies of anti-HIV treatment. Use separately PI can make viral load reduce 2-3 the order of magnitude, unite other antiviral drugs uses and virus levels can be reduced to the level that can't detect. Using the problem of PI at present is such as side effects such as hyperlipidemias. Another serious problem is to use for a long time PI because of its viral multidrug resistance. The something PI of these resistant mutations is specific, but a plurality of sudden changes gatherings of patient's protease cause majority even all PI resistances to the action of a drug (Palmer, Shafer and Merigan, 1999:Vickrey etc., 2003) in the PI therapeutic process. It is higher that cross tolerance produces the frequency ratio expection, causes any obtainable PI to treat unsuccessfully (Palmer, Shafer and Merigan, 1999). For example, MDR769 protease is to separate from a failed patient for the treatment of, except indinavir being had 14 times resistance, all granted PI is all had 40 times resistance (Logsdon etc., 2004). The generation of protease Multidrug resistance is that PI infects the main cause that finally loses result for the treatment of to HIV in these therapeutic processes, has produced HIV multidrug resistance growth rate and has surpassed at present obtainable PI quantity. Therefore, need badly for the new active PI of resistance to the action of a drug protease exploitation.

Therefore, the little PI of new side effect can treat HIV-1 and infect, and acts on the protease that existing PI is had resistance. The cell screening system that utilizes fission yeast to carry out new PI screening has two advantages. At first, cell system has clear superiority, and toxic chemical can not pass cell membrane and be left out, and intracellular reactive compound tests positive. Secondly, the compound CKIs enzymatic activity of screening and whether be combined with the avtive spot of enzyme with them has nothing to do. All PI of FDA approval in 2005 all are combined with the avtive spot of protease, and their exploitation depends on avtive spot CONSTRUCTED SPECIFICATION information. A kind of like this screening sequence might be found the inhibitor for other zone of protease and domain. For example, the new mechanism of a CKIs enzymatic activity is that this only has by the cell screening system and can detect for the degraded of 26S proteasome. As new synthetic non-correct unfolded protein or the unfolded protein of activated protein catabolite, often by 26S proteasome enzymolysis (Wolf and Hilt, 2004). One stops the folding compound of new synthetic proteins enzyme to be degraded by the 26S proteasome in short-term, but such compound can't detect by acellular screening system, because it does not exist new synthetic protease and proteasome. In this example, need emphasis to be pointed out that, the degradation pathway (Parodi, 1999) of a large amount of similar 26S protease is arranged in fission yeast and human body cell, this similarity contains many other elementary cell process (Zhao and Elder, 2000; Zhao and Lieberman, 1995). Thereby compound needs the cytoactive process, and the fission yeast cell is one and well develops the screening compound system that cell is the basis that the compound that filters out can be used for the mankind.

At this, the inventor has developed a fission yeast system, makes cell death by inducing the HIV-1 protease gene to express. Except affecting Growth of Cells and activity, this invention provides a test of measuring the yeast cells endoprotease activity, and the reorientation that this test can utilize the green fluorescence of GFP to carry out from nucleus to whole cell scope shows proteinase activity. PI indinavir protease inhibition in cell, this can be by the effect of its cell growth and the reorientation test reflection of GFP. Three of the fission yeast system show that reading can be used for exploitation high flux screening (HTS) system. In addition, utilize sudden change, the protease resistant gene is used for the HTS system makes the compound of screening that broad spectrum activity for the protease of wildness and sudden change be arranged. The compound of Screening and Identification can be developed as the resistance protein enzyme of finding among the patient of present therapeutic scheme failure like this.

At least some aspect of the present invention has considered that the inhibitor of antiprotease comprises, for example for HIV-1 protease or HIV-2 protease, perhaps simultaneously for both. In addition, the fission yeast system allows fast high-flux screening antiprotease reagent in cell. Because it is inherent different that this newly-established cell system and traditional structural design have, and uses the anti-PR medicine of this new screening system, the scheme that provides at least a kind of anti-HIV to treat.

III.HIV-1 protease

Protease is a kind of enzyme that protein is cut into the component polypeptide. Produce the activated protein composition of ripe virus after " multimeric protein " is sheared, these albumen consist of human immunodeficiency virus (HIV). HIV-1 protease is an aspartic protease of shearing newborn polyprotein in the virus replication.

The viral polyprotein of HIV-1 protease (PR) hydrolysis forms the functional protein product to virus assembling and active tool important function, can promote virus to sprout from host cell and form the maturation of virion. PR is homodimer, and each monomer is by 99 Amino acid profiles, conformation too, the N end is proline Pro, the C end is phenylalanine Phe. Each monomer position of active protease arrangement that is centrosymmetric, its secondary structure comprises an alpha-helix and two antiparallel β lamellas.

Except relying on noncovalent interaction to stablize the dimer, catalytic residue, the aliphatic amino acid residue of the hydrophobic interaction of side chain and each monomer hydrophobic core have played important function. Each monomer comprises two cysteine residues, but does not participate in forming disulfide bond. Form avtive spot at the dimer interface place, form by the crack that produces between two domains as four β-pleated sheet parts back and forth, its position is in the center near molecule. Avtive spot is covered by the corner of an expansion, a β hair fastener ring and a β lamella. This laminated structure is from 46 phenylalanine (Met) until 55 lysine (Lys), has flexibility like the hinge-like motion, the end that enters hydrophobic region by switch promotes that substrate reaches active site, brings into play the central role of PR activity with this. The PR ovalize is also relatively flat, and several potential calmodulin binding domain CaMs are arranged in the crack of hollow.

The shearing of the enzymatic protein chain of albumen is 25 two asparatates (Asp-25) mediations arranged side by side of avtive spot, and these two asparatates form one " catalytic dimerization body ". It is 3.3 that two one of them pKa values of carboxyl are lower than ordinary, and another is 5.3 of routine. This catalytic dimerization body often interacts with acylamino-and is sheared into peptide substrate.

Under concrete situations more of the present invention, HIV-1 protease refers to human HIV-1 protease, and further HIV-1 protease polymerized nucleoside acid sequence refers to SEQ ID NO:1 sequence, and peptide sequence refers to SEQ ID NO:2 sequence. Equally, under concrete situations more of the present invention, HIV-2 protease refers to human HIV-2 protease, HIV-2 protease polypeptide sequence refers to SEQ ID NO:5 (GenBank Accession No. 2MIPC, gi:443404) sequence, HIV-2 protease polymerized nucleoside acid sequence refers to SEQ ID NO:6 sequence. Under other situations, Accession No. S42993 among the database GenBank of NCBI, gi:354697; The HIV-1 virus infections factor (vif) of SEQ ID NO:7 also is employed. In replacement scheme, the separately sequence that provides from here, HIV-1 protease or HIV-2 protease have the variation of one or more sequences, need only protease at the yeast cells overexpression, and cell will be dead. Used sequence and sequence provided herein have 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% uniformity at least among the present invention.

Present HIV-1 protease inhibitors comprises, for example Invirase (saquinavir mesilate, saquinavir mesylate; Its chemistry N-tert-butyl group by name-D hydrogenation-2-[2 (R)-hydroxyl-4-benzyl-3 (S)-[[N-(2-quinolyl formoxyl)-D-asparagine] amino] butyl]-(4AS, 8AS)-isoquinolin-3 (S)-C formyl ammonia methanesulfonic acid;

N-tert-butyl-decahydro-2-[2 (R)-hydroxy-4-phenyl-3 (S)-[[N-(2-quinolylcarbo nyl)-D-asparaginyl] amino] butyl]-(4aS, 8aS)-and isoquinoline-3 (S)-carboxamide), molecular formula is C₃₈H ₅₀N ₆O ₅.CH ₄O ₃S, molecular weight are 766.96, wherein the molecular weight 670.86 of free radical; These inhibitor are in U.S. Patent No. 6,043, narration are arranged in 357; Legal invention number of registration is H1649; Also comprise such as Ritonavir (Ritonavir, Norvir), VX-478 (Amprenavir), atazanavir (Atazanavir, ATV), husky that Wei of Puli (Fosamprenavir) of good fortune, indinavir (Indinavir), Lopinavir (Lopinavir), tipranavir (Tipranavir), NELFINAVIR (Nelfinavir, Viracept) etc. The protease inhibitors that the present invention differentiates can be used for replacing one or more at present known HIV-1 protease inhibitors, perhaps replenishing as existing protease inhibitors.

IV.HIV-1 proteinase inhibitor drug candidate

Aspect some, the screening method of a HIV-1 proteinase inhibitor of evaluation of use is directly to differentiate the HIV-1 protease inhibitor of the present invention.So long as can suppress the death of overexpression HIV-1 proteolytic enzyme yeast cell, these inhibitor can be any kinds.In some alternative methods, can differentiate more weak inhibitor, for example pass through the slower bacterium colony of selection growth at least once.

Under particular condition, the HIV-1 proteinase inhibitor can be one or more albumen, polypeptide, peptide section, antibody, DNA and/or RNA oligonucleotide, small molecules, synthetic compound, natural compounds etc.Inhibitor also can screen from other similar molecular libraries, for example, can utilize drug candidate setter library, comprises multiple organism, as schizosaccharomyces pombe and yeast saccharomyces cerevisiae, the mankind, mouse, mouse, fungi, fruit bat, nematode, Arabidopis thaliana, Fugurubripes etc.Certainly, sometimes also substitute with the small molecules library.An example is to collect molecule from a strain or many strains plant, and used typical plant also comprises the medicinal herbs of China in the library.The library also comprises RNA interfering, for example comprises RNAi, siRNA and shRNA.In some aspects, the small molecules literary composition comprises the HIV-1 and/or the HIV-2 inhibitor of inhibition based on known protein enzyme inhibitors structure choice.

V. yeast

Yeast is unicellular fungi, and cell regulate and control mechanism is very similar with the human cell, and its precise classification is a field that depends on cell, spore and colony morphology characteristic, also comprises physiologic character.Another more notable attribute be that sugar-fermenting is produced the alcoholic acid ability.Budding yeast belongs to the Ascomycota Hemiascomycetes, and the eucaryon yeast is divided into two main Saccharomycetaleses.The assorted distributed in nature of yeast is extensive, finds in be everlasting leaf and flower, soil and salt solution, also deposits as a kind of symbiosis or parasitic mode and is skin surface homoiothermous and internal organ.

The mode of yeast single cell breeding comprises sprout (for example Saccharomycetes) or amitosis (fragmentation, for example fission yeast bacterium), and perhaps they are simply grown with irregular thread (mycelium).Great majority form in the yeast syngenesis mode of ascus, comprise the single spore more than eight at least, and these spores can be with nuclear fusion and by nutrition division multiplication, and perhaps some yeast can merge with other spore.

The yeast cell that this invention is used is not limited only to Saccharomycetes (yeast saccharomyces cerevisiae for example; Cereuisiae fermentum), fission yeast (as schizosaccharomyces pombe), pichia (as pichia pastoris phaff), saccharomyces hansenii (as the methylotrophy yeast), kluyveromyces (as Kluyveromyces lactis) and Ye Shi yeast (as separating fat Ye Shi yeast).

What some particular aspects of the present invention was used is schizosaccharomyces pombe.The QuickDraw ability of part of developing of yeast genetics owing to schizosaccharomyces pombe genome phenotype generation gene region.Schizosaccharomyces pombe has become the modular system of many molecule genetics research.

A. yeast cell is cultivated

Some yeast specie reproduction speeds are the same with bacterium fast, and the genome size is only less than mammiferous 1%.They can be carried out fast the molecular genetics operation, and its gene can be deleted, replace or change.They also have unconventional monoploid propagation mode, and each gene has only single copy in the cell, and this makes the inactivation that separates and study certain gene become easily, has also avoided the complicacy of another gene copy of cell domestic trouble.

Saccharomycetic cultivation comprises separation, yeast culture and the zymic breeding of single yeast cell, up to the culture that obtains sufficient amount.Pure yeast culture source is a lot, for example can buy commercially produced product or pass through to cultivate collection.Collecting pure growth has various flow processs, comprises from the mixture of single bacterium colony, individual cells or isolated cell and bacterium colony beginning to cultivate.

The target of breeding is to obtain a large amount of clear and definite yeast of performance in the short as far as possible time, a kind of method is a propagating system in batch, promptly utilize several milliliters cultivation progressively to amplify up to the yeast that obtains aequum, adding active strong grown cell in the amplification process provides fresh nutrition, yeast is copied under the physiological status of the best duplicate.

B. Yeast promoter

In some aspects, a regulation and control zone refers to a promotor, is used to promote the expression of hiv protease and makes necrocytosis.Promotor may be positioned on the plasmid (or other carriers) relevant with the operation of proteolytic enzyme polynucleotide, also may be positioned at the yeast genes group.Promotor can be an overexpression, and is derivable or the constructive expression.

Therefore, the expression structure that comprises HIV-1 proteolytic enzyme contains a regulating and controlling sequence, and the expression of regulating HIV-1 proteolytic enzyme is to the level of impelling necrocytosis.For example, regulating and controlling sequence may impel HIV-1 proteolytic enzyme overexpression and the kill yeast cell.Under other situations, regulating and controlling sequence can be induced, for example by directly inducing the expression of HIV-1 proteolytic enzyme and being exposed in the specific substratum.Gal 1 and Gal 10 are these class promotors, can induce by semi-lactosi.Changing expression by opening or closing of such gene often is worth, because this makes the researchist to regulate expression according to the time.

Under some situations, yeast cell is the fission yeast cell, the typical promotor of fission yeast comprises adh1+ (composition high expression level), and fbp1+ (carbon source induced reaction) suppresses the system and nmt1+ (no signal in the thiamines) promotor of frequent use at present based on the tsiklomitsin of CaMV promotor.

The nmt1+ promotor has three versions: all one's effort promotor and two versions that all weaken under inhibition and inductive condition.A guerrilla warfare different polylinker in the REP/RIP of nmt carrier series can partly activate by the concentration of regulating thiamines.Fully induce: no thiamines.Fully suppress: the thiamines of 20 μ M (5 μ g/ml).Part is induced (as described in reference): 0.05 μ M thiamines (0.016 μ g/ml).

The nmt1 promotor can not closed fully, and the ability that makes up " cut-out " plasmid greatly depends on by the sensitivity to specific protein dosage of expressed protein and cell.Even the gene that many nmt1 control is expressed also can be replenished by the most weak promotor under the situation that thiamines exists, but the example that also has many genes successfully to be closed to obtain the nonsense phenotype.Therefore, must determine that how utilizing this class promotor to carry out plasmid closes experiment according to each gene particular case is experimental.

Other expresses nmt1, fbp1+, inv1+ and the ctr4+ that useful promotor comprises that direct promotion is expressed to conditionality, can think that these promotors are very useful to schizosaccharomyces pombe.Other promotor, very useful to yeast saccharomyces cerevisiae, for example comprise metallothionein(MT), glycerol 3-phosphate kinases, such as I type carbohydrate-splitting enzyme and other enzymes of Hydratase, phosphoenolpyruvate or glyceraldehyde-3-phosphate dehydrogenase etc., wherein glyceraldehyde-3-phosphate dehydrogenase is responsible for the utilization of maltose and semi-lactosi.Other stronger Yeast promoter is ethanol dehydrogenase, Sumylact L and I type triosephosphate isomerase promotor.

Gal 1 gene is adjacent with Gal 10 genes, transcribes with opposite direction from same promoter region.The regulation and control zone that contains the UAS sequence can be cut off at DedI Sau3A fragment place, and any one other gene of placed upstream can promote the semi-lactosi abduction delivering and suppress lactose and express.Composition high expression level promotor when PGK, GPD and ADH1 promotor (PGK=phosphoglycerokinase, GPD=glyceraldehyde 3 phosphate desaturase, ADH1=ethanol dehydrogenase).The ADH2 promotor is that a glucose suppresses promotor, transcribes strongly not induced by semi-lactosi in non-fermented type carbon source (similar with GAL 1 or 10).CUP1 promotor metallothionein promoter activates in substratum by adding copper or silver element, also be in the yeast cell genome copy number more than one in several genes of one.According to the difference of bacterial strain, this gene can be up to eight copies.The PHO5 promotor is the secretory dna of coding acid phosphatase, and at lower concentration or do not have in the substratum of phosphoric acid and induced, the secretion of Phosphoric acid esterase can impel some phosphoric acid to discharge from surrounding environment.The PHO5 no signal is arranged under the situation of phosphoric acid; During no phosphoric acid, it presents strongly.

C. yeast conversion flow process

There are many means can transformed yeast cell, comprise electric commentaries on classics, Lithium Acetate and protoplastis.Under a kind of situation, a molecule can be by simply placing the substratum that contains this molecule to enter yeast cell in yeast.In some situation of the present invention, molecule is transported in organoid, cell, tissue or the body by the electricity conversion.When electricity changes, the suspension of cell and DNA is placed high-voltage electric field.The modification of this method uses the cell wall degrading enzyme as the polygalacturonase to handle, and the target cell that makes processing transforms responsive more (United States Patent (USP): U.S.Patent No.5,384,253, the document part sees reference) than untreated cell to electricity.Zymolase can be used on the schizosaccharomyces pombe at least.Physical abuse can make competence more responsive to transforming.

Protoplasma merges the sex obstacle (Svoboda, 1976) that has been used to overcome the hereditary onrelevant strain mating of prevention, and this helps genetic constitution exchange (Provost etc., 1978 in whole or in part; Wilson etc., 1982; Perez etc., 1984; Spencer etc., 1985; Pina etc., 1986; Skala etc., 1988; Janderova etc., 1990; Gupthar, 1992; Moluar and Sipiczki, 1993).This process depends on the digestion of cell walls, and the protoplasma that merges (Kao and Michayluk, 1974) and be used for the yeast fusion test such as polyoxyethylene glycol subsequently adsorbs promotor, Ca2+ (vanSolingen and Van der Plaat, 1997; Svoboda, 198; Wison etc., 1982; Pina etc., 1986).Other staff make the electricity consumption integration technology substitute polyoxyethylene glycol, have reported " a kind of enhanced protoplasma fusion rate " (Weber etc., 1981; Halfrnann etc., 1982).The effect of polyoxyethylene glycol and indeterminate, the aggegation of the of the same race or heteroplasm of its catalysis.

Fusion process is summarized as follows: (1) protoplasma is fused into the grumeleuse of all size at random; (2), change grumeleuse into synplasm (" chimeric protoplasma fusion product ") (Ahkong etc., 1975a by the disintegration of film and incorporating into of protoplasma content; Gumpert, 1980; Svoboda, 1981; Klinner and Bottcher, 1984); (3) structure of film (Ahkong etc., 1975a; Gumpert, 1980) and fusion (Sarachek and Rhoads, 1981 of heterokaryon kernel; Klinner and Bottcher, 1984).

Another kind method is to utilize electroporation.At first use minimum medium (arriving the stationary phase (OD595=1.5) early of the transformation frequency influence of not grown before) to make cell grow to about 1 * 107/ml (OD595=0.5).Collecting cell, 20 ℃, centrifugal 5 minutes of 3000rpm then utilizes the sorbyl alcohol washing of the good 1M of ice bath after the water washing once of using ice to handle.Existing report that these cells are hatched at the DTT of 25mM (dithiothreitol (DTT)) and can strengthen the electroporation ability in 15 minutes.Cell is resuspended in the sorbyl alcohol of 1M of ice bath the most at last, and density is 1-5 * 109/ml.The suspension of 40 μ l cell I joined in the pipe of precooling of the DNA (100ng) that contains conversion ice bath 5 minutes, and the electroporation operation parameter is provided with as follows: (a) 1.5kV, 200ohms, 25 μ F (Biorad); (b) 1.5kV, 132ohms, 40 μ F (Gensen/Flowgen).Cell and DNA transfer to the Glass tubing afterpulse of a precooling and handle; 0.9ml the 1M sorbyl alcohol of precooling adds Glass tubing immediately; When other electroporation carries out with ice bath in the cell suspension return tube.Fast as far as possible cell is layered on selected on the flat board, cultivates that transformant will begin to occur after 4-6 days for 32 ℃.

Following Lithium Acetate draft is from (1990) such as Okazaki, high frequency method for transformation and pass through the Mammals cDNA clone's of schizosaccharomyces pombe reverse complemental library transduction vector.It is 0.5-1 * 107 cell/ml (OD595=0.2-0.5) to density that cell grows in the 150ml minimum medium.Cell growth in the substratum that contains low concentration glucose or MB substratum (with reference to Okazaki etc.) is not fine, but can improve transformation efficiency.3000rpm is after centrifugal 5 minutes, with last time the same centrifugation together again after the aseptic washing of 40ml under the collecting cell room temperature.Concentration with 1 * 109 cell/ml is resuspended to (acetic acid is regulated pH to 4.9) in the 0.1M Lithium Acetate solution, and is divided into a plurality of 100ul adding EPPendorf pipes, and 30 ℃ (the ts mutant is 25 ℃) hatched 60-120 minute.Every pipe adds the 1 μ g plasmid DNA that is dissolved in 15ul TE (pH7.5), and slight the mixing, makes sedimentary cell resuspension in the process of hatching.Must keep the temperature of pipe not descend during this period.The PEG 4000 (w/v) of 290ul 50% is preheating to 30 ℃ (the ts mutant is 25 ℃) back and adds in the pipe, and then slight mixing back 30 ℃ (the ts mutant is 25 ℃) was hatched 60 minutes.15 minutes postcooling of 43 ℃ of heat shocks of these pipes used Eppendorf whizzer 5000rpm centrifugal 2 minutes to room temperature, careful sucking-off supernatant liquor in 10 minutes.Utilize transfer pipet piletman P1000 to suck sucking-off repeatedly, cell is resuspended to the 1/2YE broth culture, transfer to then in the 50ml flask, add the 1/2YE dilution of 9ml.Cell was cultivated 60 minutes or the longer time in 32 ℃ (the ts mutant is 25 ℃) concussion, got being laid on the basic culture plate less than 0.3ml of equivalent.If desired, be resuspended to 1ml substratum and sprawl more many cells to flat board behind the cell centrifugation with this period.

D. zymic codon preference

In order to obtain the peptase of yeast cell optimum expression, the Nucleotide of coding peptase will be synthetic according to the design of yeast codon preference.Following table provides the codon such as the yeast preference of schizosaccharomyces pombe etc.

Table 1: the codon preference of schizosaccharomyces pombe

Gly	GGT，GGA
		Glu	GAA
Asp	GAT
		Val	GTT
Ala	GCT
		Lys	AAA
Asn	AAT
		Ile	ATT
Thr	ACT
		Cys	TGT
End	TAA
		Tyr	TAT
Leu	TTG，TTA，CTT
		Phe	TTT
Ser	TCT

Arg	CGT
		Gln	CAA
His	CAT
		Pro	CCT

E. yeast mark

Aspect some, utilize mark to monitor the existence of special oligonucleotide of the present invention.Mark can be nutrition mark or antibiotics resistance mark, can use one or more marks.Though any nutrition mark that the oligonucleotide of certification mark exists can utilize, the main nutrition mark that uses comprises ade1, ade6, arg3, CAN1, his3, his7, leu1, leu2, sup3-5, ura3 and ura3 among the present invention.Simultaneously, though the antibiont mark that the oligonucleotide of any certification mark exists can use, the resistance sign that the present invention mainly uses mainly comprises kantlex, G418 and Totomycin.

F. Yeast expression carrier

In some cases, expression vector comprises the one or more compositions in the existing invention.Though any suitable carrier all can use, in concrete test, used one or more fission yeast carriers, as shown in table 2, also can search a wherein part by the Global Internet station of the Susan Forsberg of University of Southern California.

Table 2: typical general fission yeast carrier

VI. test detects

As mentioned above, the yeast cell system among the present invention is designed to differentiate and saves owing to cross the inhibitor of expressing the necrocytosis that HIV-1 proteolytic enzyme causes and/or differentiate the inhibitor that stops protease activity, for example cuts off protease substrate.

The a certain concrete situation of test development is optimized and is verified the test of one or more fission yeasts, is used for typical 96 orifice plates and measures, particularly comprise following some:

1) will in RE294 fission yeast bacterial strain, measure HIV-1 proteolytic enzyme induced growth suppress active agarose plate for the basis mensuration be converted into absorbance measurement.

2) the fluorescence reading of adjusting yeast survival/death is determined the necrocytosis of proteolytic enzyme inductive.

3) GFP fluorocyte reorientation test is converted into the active high-density fluorometric assay of monitoring hiv protease, this test is detected as technology screening.

In the test of HTS type, the testing method of exploitation is carried out the second phase optimize, further make up HTS, can:

1) be the HTS form with all HIV-1 proteolytic enzyme test modifications, if possible miniaturization as far as possible;

2) with SAMI NT software application to being in the laboratory fully automatic system of core with the HTS system, with control comprise fluid operated and be incubated in all test operations;

3) set up all reaction normals according to quality-controlling parameters, comprise and measure repeatability, the variation coefficient (CV value), signal to noise ratio and Z factor analysis;

4) use the active LOPAC1280 compound library of pharmacokinetics (Sigma-RBI) operation to detect.

After the adjustment of having carried out suggestion and confirming, these fission yeast cell tests will be used for the HTS screening of new HIV-1 proteinase inhibitor, and these inhibitor participate in the network at molecular library screening center.

The shear active of HIV-1 proteolytic enzyme can be measured with any suitable mode.Though in particular experiment, this measures by detecting specific HIV-1 proteolytic cleavage site.Any hiv protease site can be used, but the present invention mainly comprises following site: DSQNYPIVQ (SEQ ID NO:3); DSFNFPOIT (SEQ ID NO:4); VSQNYPIVQN (SEQ ID NO:8); KARVLAEAMS (SEQ ID NO:9); SATIMMQRGN (SWQ ID NO:10); RPGNFLQSRP (SEQ ID NO:11); VSPNFPQITL (SEQ ID NO:12); CTLNFPISPI (SEQ ID NO:13); GAETFYVDG (SEQ ID NO:14) or IRKVLFLDGI (SEQ ID NO:15) (with reference to Wlodawer and Gustchina, 2000).

Express the host cell death that heterogeneous HIV-1 proteolytic enzyme causes, but add a kind of inhibitor under the condition that helps the proteolytic enzyme expression, the necrocytosis that meeting blocks protein enzyme causes also impels the yeast cell breeding.Can directly select/differentiate the inhibitor that allows yeast cell under the proteolytic enzyme condition, to grow and/or stop protease substrate to be sheared apace by recombinant antibodies in biological polypeptide, albumen, small molecules or the cell is introduced the yeast cell that contains heterogeneous proteolytic enzyme.

A. heredity is selected

In heredity was selected, the DNA library of coding potential peptide inhibitor was transformed in the yeast cell by high frequency (every milligram of plasmid DNA surpasses 10 transformants).Transformant is cultivated on agarose plate, and this substratum contains the elicitor of proteinase gene expression inhibitor and removes an amino acid whose plasmid mark.Most of transformants can't be grown owing to the expression of proteolytic enzyme, and some cell transformed can not be grown owing to lack plasmid.Yet what contain the plasmid inhibitor in the yeast transformant can normally form bacterium colony.Can reclaim plasmid DNA according to the DNA purifying procedure of standard, suppress sub-dna sequence dna and can determine by dna sequencing.

The source of candidate inhibitor can comprise the Nucleotide library, contains genome or cDNA library, albumen library, polypeptide libraries, RNA library, antibody library, little organic molecule library.Gene can be any form, as long as they can transform and enter yeast cell.Genomic library can derive from yeast (comprising schizosaccharomyces pombe and yeast saccharomyces cerevisiae), the mankind, mouse, mouse, algae, fungi, fruit bat, Arabidopis thaliana and FUGU RUBRIPES RUBRIPES.

B. high flux screening (HTS)

Can differentiate small-molecular peptides and chemical inhibitor by the method among the present invention.Yeast cell dilution and evenly distribute to the hole that contains yeast governor substratum in, are added compound, microscopic examination or porous plate reading apparatus detection yeast cell growth situation.The existence of inhibitor can impel the cell growth, and turbidity increases in the hole.This HTS test is the standard set operation, successfully has been used to differentiate micromolecular inhibitor, and with the present invention substantial differences (H μ ghes, 2002) has been arranged.In order to overcome potential limiting factors such as cell permeability, can use specific compounds such as polymyxin (Boguslaski, 1985) strengthen cell permeability, yeast strains (the Brendel that perhaps uses cell walls to undergo mutation, 1976), the multiple drug that is subjected to that also comprises expection initiatively effluxes the cell that causes infringement.

VII. combination therapy

HIV-1 proteinase inhibitor of the present invention all is the needs at individual treatment, and the individuality of infected by HIV is for example suffered from the individuality of AIDS, perhaps easy infection HIV or easily suffer from the sensitive individual of AIDS.Though the inhibitor among the present invention is not and other drug combination therapy HIV or AIDS, in order to increase the result of treatment of inhibitor, it is necessary that these compositions and other reagent are joined together to treat HIV/AIDS.More likely be these components can the more effective HIV of killing virus and/or cellular constituent by associating.In some specified scheme, component in the invention and the associating of cocktail treatment reagent comprise itself and various HIV treatment reagent are share.For example, one or more proteinase inhibitor and one or more reverse transcriptase inhibitors are share.

This process also comprises the component of individual cells and invention and extra reagent or a plurality of factor is connected simultaneously.This can be cell and single component or the pharmacokinetics dosage combination that comprises two kinds of reagent, or with cell and two or more unique composition or dosage combinations.Simultaneously, a cell comprises the composition of invention, and another cell comprises second kind of reagent, and this associating can produce accumulation or synergistic effect in two kinds of treatments.

Treatment among the present invention can be carried out before or after other reagent treatment, and the timed interval can be from the several minutes to several weeks.Under the situation that the composition of other reagent and invention separately uses, must guarantee that the effective time cycle can not be shorter than the interval of twice administration, reagent and invention component can continue to bring into play the joint effect of pair cell like this.In this case, must consider two kinds of situations, twice administration was separated by 12-24 hour, more likely was 6-12 hour.In some cases, can significant prolongation cycle treatment time, yet, twice administration also at interval several days (2,3,4,5,6 or 7) to several weeks (1,2,3,4,5,6,7 or 8).

If the invention composition is A, other reagent is B, various associated forms can for:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Conventional flow process is followed in inhibitor component for treating of the present invention administration, and these flow processs are used for other HIV/AIDS treatment.Everybody expects the repetitive therapy cycle on demand, also can consider various standard cares and the inhibitor for treating combined utilization of describing.

Other the HIV/AIDS treatment reagent of uniting use with the present invention can be the following: non-nucleotide reverse transcriptase inhibitors, as Delavirdine, efavirenz, nevirapine; The ucleotides reverse transcriptase inhibitors is as Abacavir, lamivudine, zidovudine, stavudine, tynofovir, zalcitabine; Proteinase inhibitor is as amprenavir, Reyataz R, husky that Wei of Puli of fluorine, Indinavir, rltonavir, ritonavir, viracept see nelfinaivr, Saquinavir; And/or fusion inhibitor, as En Fuwei ground.

VIII. pharmaceutics preparation

Pharmaceutics of the present invention is formed HIV-1 and/or the HIV-2 proteinase inhibitor that comprises Screening and Identification in one or more inventions, also can add extra reagent, is dissolved or dispersed in the pharmaceutics acceptable carrier.In the concrete case for example of the present invention, the pharmaceutics component comprises hhp2 polynucleotide or polypeptide.Phrase " pharmaceutics or pharmacokinetics are acceptable " is meant that when animals administer molecular entity and component can not brought out side effect, allergy and uncomfortable reaction, like this human body are only suitable.The pharmaceutics composition comprises at least one HIV-1 proteinase inhibitor or adds active intermediate, its preparation needs current in the industry once some technical ability that disclose, as the pharmacy science of Remington (the 18th edition, Mack publishing company, 1990), these are quoted in reference.In addition, to animal (for example human) administration, its preparation need meet aseptic, the thermal source of FDA biologic criteria requirement, conventional safety and purity requirement, and this is readily appreciated that.

Making from here is used for seeing that " acceptable vehicle on the pharmaceutics " comprises any material and all solvents, as dispersion medium, and sugar-coat, tensio-active agent, sanitas (for example antibacterial agent and anti-mycotic agent), the isotropic substance preparation absorbs the retardation agent, salt, medicine, medicine stablizer, gel, tackiness agent, excipient, decomposition agent, lubricant, sweeting agent, seasonings, dyestuff is such as materials similar and compound.This is a kind of conventional technical ability (the pharmacy science of knowing in the industry of Remington: 1289-1329 page or leaf; The 18th edition, Mack publishing company, 1990, reference is quoted).At present, except being not suitable for pharmaceutical intermediate, it all is a considerable thing that any conventional carriers is used for the medicament component.

It is solid, liquid or gas that the contained different vehicle type of HIV-1 proteinase inhibitor depends on pharmaceutical dosage form, also with as route of administration such as injection whether need aseptic relevant.Medicament among the present invention can vein, in the intracutaneous, transdermal, sheath, in the intra-arterial, intraperitoneal, nose, in the leaf sheath, rectum, part, intramuscular, subcutaneous, mucous membrane, oral, local, suck (sucking), inject, inculcate as gas, transfusion continuously, local soaking target cell pour into administration, by conduit, bowel lavage, emulsion, in liquid ingredient (as liposome), or by additive method or people and ongoing ordinary method in the industry.

The HIV-1 proteinase inhibitor is free to give the form of neutrality or salts solution, salt acceptable on the pharmaceutics comprises bisalt, the free amino group of those protein ingredients for example, perhaps organic acid, hydrochloride or phosphoric acid salt are perhaps as organic acids such as acetate, oxalic acid, tartrate, mandelic acids.The salt that free carboxyl group forms can derive from non-organic elements such as sodium, potassium, ammonium, calcium or ferric oxide, or organic compositions such as Isopropylamine, Trimethylamine 99, histamine or PROCAINE HCL, PHARMA GRADE.For formulation, solution form administration mode should be corresponding with dosage, guarantees the effectively required amount of treatment.The very easily administration of this formulation comprises a series of modes, and as the parenterai administration of injection of solution class, gas is sucked into lung, perhaps as the digestive tube administration of release capsule analogue.

For further consistent with the present invention, pharmaceutical cpd of the present invention is contained in the acceptable vehicle of pharmaceutics, comprises or do not comprise inertia solution.Vehicle should be absorbable, comprises liquid, and is semi-solid as ointment, solid.Except conventional carriers up to now, reagent solution or as excipient with outside the carrier that improves curative effect, the administration that they is used for composition of the present invention also is suitable.Carrier or solution comprise fat, oil, salts solution, lipid, liposome, grease, tackiness agent, weighting agent and analogue, perhaps their associating.Also can contain the various antioxidants that delay oxidation in the component.In addition, prevent that microbial activities from can use sanitas, as utilize antibacterial agent and anti-mycotic agent, include but not limited to disinfectant (as methyl p-hydroxybenzoate, propylparaben), chlorobutanol, phenol, Sorbic Acid, sulphur mercury and their associating.

For consistent with the present invention, component and carrier are combined into any convenient and practical mode, and as solution, suspension, emulsifying agent, admixture, capsule, absorption agent and analogue, these all are quotidian in the industry.

In the concrete case of the present invention, component is and semisolid or the thorough blended of solid carrier that mixing process can be used any mode easily, as grinding.In order to protect the lost therapeutic activity of component, mixing process also adds stablizer, as the stomach sex change.The stablizer that uses in the component comprises damping fluid, amino acid such as glycine and Methionin, carbohydrate such as dextrose, seminose, semi-lactosi, fructose, lactose, sucrose, maltose, sorbose and N.F,USP MANNITOL.

In further scheme, the present invention will consider to utilize pharmaceutics lipid carrier composition, comprise the HIV-1 proteinase inhibitor, one or more lipids and a kind of aqueous solution." lipid " used herein is defined as and comprises any being not soluted in water widely and can be by the material of organic extractant solution, and these materials are known for personnel's right and wrong Changshu in the industry, and said herein " lipid " also is not limited to any special structure.For example comprise and contain long-chain fat hydrocarbon and their derivative.Lipid can spontaneous generation or synthetic (as artificial design production).Yet, lipid is the biology substrate normally, biology lipid is widely known by the people, and comprises neutral fat, phosphatide, phospho-glycerol, steroidal, terpenes, lysogeny lipid, sphingoglycolipid, glycolipid, thioester, the lipid acid that contains ether and ester and polymerization lipid, perhaps the associating between them.Certainly, except the compound that is widely known by the people that those were specifically told about, the present invention has also comprised other lipid and has constituted and method.

A conventional technical ability in this field comprises is familiar with the technology that those are distributed to component Lipid carriers.For example, the HIV-1 proteinase inhibitor disperses to enter lipid solution, is dissolved in lipid, lipid emulsification, mixes with lipid, and contaminated with lipid, covalently bound to lipid, formation lipid suspension, incapsulate or liposome in, or combine by any known way with lipid or lipid structure.This distribution perhaps causes forming liposome.

The actual dosage of drug component is determined according to the physics and the physiologic factor of infected animal, as situation, patient's sudden illness situation and the administration route of body weight, severity, disease type, original treatment and co-therapy.Different according to dosage and administration path, the route of administration quantity of preferential dosage and/or effective dose is by the reaction decision of main body.In any case, actual operator is responsible for administration, determines the concentration and the proper dosage of individual pharmaceutical intermediate.

In some cases, drug component may comprise 0.1% of an active compound, and under other situations, the active compound weight ratio can be between 2%-75%, or between 25%-60%, or any other scope.Nature, the active compound quantity of each treatment active principle can prepare according to following principle: the compound by any given unit obtains suitable dosage.In the process of preparation pharmacy formulation, should consider solvability, bioavailability, biological half time, administration route, shelf life of products and other pharmacokinetics factor.Simultaneously, different dosage and treatment plans also should be considered.

In other limiting examples, dosage can be 1 microgram/kilogram/body weight (μ g/kg/bodyweight), 5 μ g/kg/body weight, 10 μ g/kg/body weight, 50 μ g/kg/body weight, 100 μ g/kg/body weight, 200 μ g/kg/body weight, 350 μ g/kg/body weight, 500 μ g/kg/body weight, 1 mg/kg/body weight (mg/kg/body weight), 5mg/kg/bodyweight, 10mg/kg/body weight, 50mg/kg/body weight, 100mg/kg/bodyweight, 200mg/kg/body weight, 350mg/kg/body weight, 500mg/kg/bodyweight is to 1000mg/kg/body weight or more, even can be any dosage.The scope of deriving out from these quantity of listing is the dosage of 5mg/kg/body weight to 500mg/kg/body weight.

C. digestive tube pharmaceutical cpd and formulation

In the priority scheme of the present invention, the HIV-1 proteinase inhibitor is by the digestive tube administration, and all possible route of administration of the drug component of Pi Luing is directly related with digestive tube herein.Particularly, this place say drug component can be oral, oral cavity, rectum or my humble abode administration.Therefore, these components can absorb the carrier excipient by inertia solution or edible, perhaps are pressed into tablet, perhaps directly put into food.

Some situation active compound is integrated into excipient to be become and can digest tablet, buccal, tablet, capsule, elixir, suspension, syrup, the aqueous solution and analogue (Mathiowitz etc., 1997; Hwang etc., 1998; United States Patent (USP) U.S.Pat.Nos.5,641,515; 5,580,579 and 5,792,451.Each is all quoted by reference).Tablet, tablet, pill, capsule analogue can comprise following material: tackiness agent, as tragacanth, gum arabic, W-Gum, gelatin or the associating between them; Excipient is as Lin Suanergai, N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate or the associating between them; Decomposition agent is as W-Gum, potato starch, sodium alginate or the associating between them; Lubricant is as Magnesium Stearate; Sweeting agent is as sucrose, lactose, asccharin or Magnesium Stearate; Seasonings is as mint, wintergreen oil, cherry seasonings, orange seasonings etc.When dose unit is capsule,, also comprise liquid vehicle except above-mentioned substance.Various other materials can wrap by or the physical form of modifying dose unit, for example tablet, pill or capsule can use sheet glue, sugar or both to share parcel.Capsule also comprises liquid vehicle except above-mentioned substance.Gelatine capsule, tablet or pill can add the enteron aisle sugar-coat.The medicine sex change that the enteron aisle sugar-coat stops stomach and upper intestines sour environment to cause is with reference to United States Patent (USP) U.S.Pat.No.5,629,001.Behind intestine and small intestine, internal pH-values is impelled dressing to dissociate to discharge pharmaceutical cpd, and is absorbed by specific cells, as enteron aisle endotheliocyte and enteron aisle lymph aggregated lymphatic follicles cell (Peyer ' spatch cells).Syrup can comprise sucrose as methylating reagent, adds propylparaben as sanitas, also comprises dyestuff and seasonings, as cherry and orange seasonings.Certainly, any material as pharmaceutical dosage form all should satisfy pharmacopedics purity, does not also have the toxicity of essence simultaneously.In addition, active compound can incorporate in the formulation that continues to discharge.

The oral administration of the bright component of this law can selectivity be integrated into one or more excipient, as collutory, toothpaste, orally administering, tablet, oral sprays or hypogloeeis oral administered dosage form.For example, collutory can be put into suitable solvent with the active intermediate of aequum, as dobell's solution.Perhaps active intermediate can place oral liquid, as Sodium Tetraborate, glycerine and potassium borate, or is scattered in the toothpaste, or adds effective dose in the component that contains aqueous adhesive, abrasive, seasonings, soaking agent and wetting agent.Perhaps component is packed into tablet or place the solution of sublingual administration perhaps is dissolved in the mouth.

Other is applicable to that the formulation of enterally administering comprises bolt match agent.Bolt match agent is a solid preparation, and different shape and weight are arranged, and normally inserts rectal administration.After the insertion, bolt match agent deliquescing, thawing/dissolving discharges active liquid.Usually, bolt match agent can comprise 0.5%-10%, more is the active intermediate medicine of 1%-20%.

D. non-enteron aisle pharmaceutical cpd and formulation

Further, the HIV-1 proteinase inhibitor can be the parenterai administration form.Herein, " non-enteron aisle " comprises that the gastric bypass administration enters.Particularly, herein indication medicament component can by but be not limited only to following mode: vein, intracutaneous, intramuscular, artery, interior, subcutaneous, the intraperitoneal of sheath, United States Patent (USP) U.S.Pat.No.6,7537,5146,613,308; 5,466,4685,543,158; 5,641,515 and 5,399,363 (each concrete case is quoted fully by reference).

Free radical or pharmaceutics acceptable salt class active compounds solution can prepare as tensio-active agents such as hydroxypropylcelluloses by adding in water.Dispersion agent also can prepare in glycerine, liquid macrogol (PEG) or both mixed solutions and oil.Under the storage and working conditions of routine, these preparation process comprise sanitas to stop microorganism growth.The medicine type that is suitable for injecting comprises sterile water solution or dispersion liquid and is used for preparing sterilized powder (United States Patent (USP) U.S.Pat.No.5,466,468 of aseptic parenteral solution or dispersion liquid temporarily; Quote fully by reference).In all situations, formulation must be a liquid form aseptic and for being easy to inject, must be stable under preparation and condition of storage, must prevent microbial contaminations such as bacterium and fungi.That carrier should dissolve in or be scattered in is moisture, ethanol, pure base polymer (as PEG, liquid PEG and analogue etc.) and suitable mixture, and/or vegetables oil.Keep the flowability of appropriateness, for example, utilize dressing, Yelkin TTS, in dispersion process, keep granular size by tensio-active agent.Add various antibacterial agents and anti-mycotic agent and stop the activity of microorganism, as this manthanoate of hydroxyl, chlorobutanol, phenol, Sorbic Acid, sulphur mercury and analogue.Under many situations, also tend to comprise the isotropic substance preparation, as carbohydrate or mercury chloride.By the reagent component delayed absorption, can prolong the soak time of injected component, as aluminum monostearate and gelatin.

For the parenterai administration of aqueous solution, for example, solution is necessary for suitable damping fluid under the necessary situation, and the salt of use capacity or glucose maintenance etc. are oozed.These special aqueous solution extremely are suitable in vein, intramuscular, the sheath and the intraperitoneal administration.Under this situation, sterile water solution is the medium that often uses in this field.For example, a doses is dissolved in 1ml isotonic sodium chloride (NaCl) solution, adds the subcutaneous position of fusion drug administration by injection of inculcating or advising of 1000ml.According to the injection for curing condition, can change dosage, for human body, medicament should meet aseptic, the thermal source of FDA biologic criteria requirement, conventional safety and purity.

An amount of active compound is dissolved in an amount of solvent, mixes, then filtration sterilization, preparation aseptic parenteral solution with above-mentioned other intermediate of enumerating.Usually various sterile active intermediates are placed the sterile carrier of basic dispersion medium, and need other cited intermediate of front.Be used for preparing the sterilized powder of injection liquid temporarily, use vacuum-drying and lyophilize preparation, produce from aforesaid other various pharmaceutical intermediates.Powdery components and liquid vehicle (as water or salts solution) are relevant, can comprise stablizer, also can not have stablizer.

E. miscellaneous pharmaceutics composition and formulation

In other suggested design of the present invention, active HIV-1 proteinase inhibitor can be designed to by each specific admixture route administration, topical (as transdermal) for example, mucosa delivery (in the nose, vagina etc.) and/or inhalation.

The component of topical comprises medical science available active compound, as ointment, paste, cream or powder.Ointment comprises the water-soluble components of all finishes, absorption agent, emulsion and topical.And cream or lotion include only emulsion.Local administration preparation comprises and is beneficial to the penetration enhancers that active intermediate passes skin absorption.Suitable toughener has glycerine, ethanol, methyl-sulphoxide, pyrrolidone and azone.The possible basal component of topical comprises PEG, lanolin, cool butter and Vaseline, also has other suitable absorption agent, emulsion or water-soluble finish.Topical formulations also comprises emulsifying agent, gel preparation and necessary anti-microbial preservative, to keep active intermediate, provides the homology mixture.Transdermal administration among the present invention also comprises use " paster ", for example provides one or more active compounds by paster, continues medication with predetermined proportion in the set time.

In some cases, the medicament component can be passed through to spray in eye drop, the nose, suck and/or other gas transmission carrier administration.Component is by the jet lung that directly is transported in the nose, with reference to United States Patent (USP) U.S.Pat.Nos.5, and 756,353 and 5,804,212 (each concrete case is quoted fully by reference).Similarly, particulate fat (Takenaga etc., 1998) and phosphoglyceride in the use nose (United States Patent (USP) U.S.Pat.No.5,725,871, quote fully by reference) also be very common in the industry mode.Equally, see through mucosa delivery and carry medicine also at United States Patent (USP) U.S.Pat.No.5, described for 780,045 kinds with the tetrafluoroethylene supported matrix.

Gas form refers to the colloid system that solid particulate can well be disperseed to enter in liquid or the pressurized gas.The typical gas preparation that sucks is by liquid propellant or liquid propellant mixed solution and suitable solvent among the present invention.Suitable propelling agent has carbohydrate and carbohydrate ethers.Container requires different according to propellant pressure, the administration of gas preparation is different according to age, body weight, severity and the response feature of main body.

IX. the representative test kit in inventing

Any component described herein all can be formed a test kit.In non-limitative example, a kind of test kit can comprise the HIV-1 proteinase inhibitor of screening method discriminating among the present invention and extra reagent.Test kit is made up of HIV-1 proteinase inhibitor and other random reagent in proper container.

Test kit comprises suitable liquid HIV-1 proteinase inhibitor, can be liquid water medium or lyophilised state.Container comprises medicine bottle, test tube, flask, vial, syringe or other container usually at least, and component can place internal tank, the preferably liquid form.Test kit uses second, third or more containers separately to adorn unnecessary component during more than a kind of component usually.Yet the associating of different components also can place in the same medicine bottle.Test kit of the present invention also comprises the mode that the binding of HIV-1 proteinase inhibitor and other reagent packing box is sold, and these packaging vessels comprise syringe or blow and melt plastic containers, can keep the activity of internal component.

When reagent constituents was one or more solution, solution can be aqueous solution, preferably aseptic aqueous solution.HIV-1 proteinase inhibitor component also can be the injection component.Under this situation, packaging vessel itself can be syringe, pipettor and/or other allied equipment, makes formulation can be used for the body sensitizing range, the injection animal, and/or even can mix use with other composition of test kit.

Yet reagent constituents may be a dry powder form.When reagent and/or component are dry powder, can make dry powder reconstruct by adding appropriate solvent.The mode that provides simultaneously in another container is provided solvent also is very common.

No matter how are number of containers and/or type, test kit of the present invention also can comprise and/or be packaged in a kind of device, and this device is used for helping to place in the animal body with HIV-1 proteinase inhibitor component drug administration by injection and/or with component.This class device can be the tool for transmitting of syringe, pipettor, forcep and/or any this class medical treatment permission.

Specific embodiment

The specific embodiment of listing below is for embodiment preferred of the present invention is described, but is not limited to these embodiment.Those skilled in the art are according to technology contents cited in the specific embodiment, it is the improved technology contents that the contriver makes on the basis of existing technology, can realize the present invention well, therefore, these technology contents have been formed the preferred mode of the present invention of implementing.Yet; those skilled in the art is according to the content of the present patent application file; can on the basis of cited specific embodiment, make some and change, and can on the basis that does not break away from invention spirit of the present invention and protection domain, obtain some similar or be equal to deformation programs.

Embodiment 1

Standard implementation mode of the present invention

The HIV-1 proteinase gene that obtains from plasmid nmt1 promotor can kill fission yeast after expressing, and is the content that we here discuss.This ability of killing fission yeast is based on the screening to protease inhibitor, also is a special field of this invention.Although it is to be proved first that the expression by proteinase gene in the plasmid comes the method for kill yeast, because with respect to a gene integration in karyomit(e), the easier operation of transfection plasmid, certainly in some aspects, carrying out the inhibition screening by the self-replacation of plasmid is not best selection.Compare with karyomit(e), the plasmid in the fission yeast is extremely unstable, gene integration is had the advantage of intrinsic stability in the karyomit(e).Gene integration can cause that more homogeneous expresses, because the plasmid replication multiple does not doubly wait from 0-30, causes in the plasmid gene expression dose rangeability bigger.

Be different from plasmid gene, utilizing integrator gene is flatly to express by homogeneous as the clear superiority of supressor screening, and the ability of potential enhanced stability.Yet it in fact is to cause one of unsettled reason (Keeney and Boeke, 1994) that integrator gene enters chromosomal standard operating instructions.The utilization standard operating instructions, during to karyomit(e), normally in the ura4 position, the ura4 regional gene of integrator gene both sides can duplicate (Fig. 1) with the homologous recombination gene integration.When the homologous recombination gene replication, be easy to occur the phenomenon of integrator gene disappearance.Therefore, the utilization standard operating instructions when screening protease inhibitor from integrator gene, occur the very high proteolytic enzyme factor sometimes and lack plastidogenetic bacterium colony background values.

The typical nmt1 promotor of energy high level expression proteinase gene can increase the probability of genetically deficient.The nmt1 promotor is subjected to the regulation and control of VITMAIN B1, but does not react the situation of VITMAIN B1.When having the vitamins B of high density in the medium, can suppress the expression of promotor.Opposite when lacking vitamins B in the cell then promotor just express.Cell absorbs vitamin storage B from surrounding medium after, place no vitamins B medium can not induce reaction it at once.Have only by cell fission to make cell interior vitamins B concentration effectively after the dilution, just can cause the high level expression of nmt1 promotor, and at least by four cell fission (Tommasino and Maundrell, 1991).

Before the nmt1 promotor works in inducing medium, require cell to carry out fissiparity, mean that individual cells can grow up to a miniature colony.When carrying out the supressor screening, if a cell in the colony loses gene, these cells just may have grown into the background colony.In actually operating, cultivate and induce the ratio that the cytogene in the medium loses and finally to cause losing of whole bacterium colony gene up to 2% sometimes.The background bacterium colony of this high density can disturb the identification of supressor.

For energy effective recognition supressor, we have worked out a very typical method, and it can detect the genetically deficient bacterium colony quickly and easily.In this method, we make it the kan gene integration link to each other (Fig. 2) with proteinase gene in karyomit(e), and the kan gene can make fission yeast resist the continued growth of G418 microbiotic in being rich in the medium of YE.The kan gene integration that designs is entered near the nmt1 gene location, be chosen in and resist the antibiotic transformant of G418 (Fig. 3) in the YE medium, the bacterial strain of all the other expressing protein enzyme genes whole apoptosis in inducing medium.Compare normally used ura4+ and leu1+ gene (Keeney and Boeke, 1994), use an advantage of kan gene to be, two genetic markers of ura4+ and leu1+ still can be selected to use when cultivation contains the bacterial strain of integral protein enzyme gene.In most of laboratory, the experimenter generally uses ura4+ or two marker gene of leu1+ to select transformant when making up the fission yeast carrier, and these two kinds of factors can both be transfected to the laggard row filter of bacterial strain.Yet the sharpest edges of utilizing kan genescreen bacterial strain are also can lose the kan gene when losing proteinase gene after the homologous recombination, thereby the G418 microbiotic is produced responsive (Fig. 3).The G418 that utilizes that will describe below carries out in the process of resistance screening, induces the background bacterium colony of proteinase gene disappearance in the medium to reduce to below 1/105.The frequency that successfully filters out supressor is very low, and therefore the background bacterium colony being reduced to low-level is a crucial step.

Illustration 2

Characteristic description with integral protein enzyme gene bacterium colony

We have carried out isolation identification to two anti-G418 transformants that are integrated with proteinase gene.The toxic action of proteinase gene high expression level can be explained from three aspects.At first, they are incubated at nmt1 promotor inhibition (+T) or inducibility (in liquid medium T) time, growth rate is acted normally in the inhibition medium, and in the inducibility medium, the growth of two kinds of bacterial strains all is suppressed (Fig. 4).Secondly, will in the inhibition medium, place inhibition or inducibility medium to cultivate again by the cell of incubation growth, and find in the inhibition medium, to form the bacterium colony of normal size, and form or form minimum bacterium colony (Fig. 5) in the inducibility medium hardly.The 3rd, place inducibility or inhibition substratum to cultivate again in the cell of growing in the inhibition medium, measure cell colony with different time points and form ability.In inhibition medium substratum, each measurement all has the cell greater than 95% to form bacterium colony, and the ability that cell forms colony in the inducibility medium descends rapidly, and in switching to 24 hours of inducibility matrix, colony forms ability drop to less than 1%.Because the nmt1 promotor occurs the strongest effect (Maundrell, 1993) of inducing during to 14-16 hour about cell cultures greatly, after proteinase gene was expressed and the several hrs of maximum effect occurred, cell had just lost viability.

The protease activity of testing right yeast colony at body that carries out after green fluorescent protein (GFP) and the HIV-1Vpr protein binding has been carried out verifying (Fig. 7).Green fluorescent protein itself can arrive each position of fission yeast cell.After GFP and the fusion of Vpr albumen, this fusions arrives the position of cell then according to the proteic signal of Vpr, found that most of melts all accumulates near the nuclear membrane of cell centre in the mode of endless belt or point, other positions almost do not have green fluorescent substance (Fig. 9 (Chen to occur, Elder etc., 1999)).The committed step of enzymic activity test is that the proteolytic enzyme cutting site between GFP and Vpr albumen imports a polypeptide linker (Fig. 7).If this proteolytic enzyme does not have activity, so by GFP, the egg white mixture that polypeptide linker and Vpr form can arrive near the nuclear membrane according to the signal of Vpr.On the contrary, if this proteolytic enzyme has activity, the polypeptide linker will be sheared so, can be distributed in each position of cell after GFP can not effectively link with Vpr.

This special linker is understood that at the body test card two kinds of bacterium colonies that have integrator gene all have protease activity by enzymolysis.Two control experiments have provided expected result.First test is any one position (Fig. 8) that influence that whether GFP is not existed by proteolytic enzyme can be positioned cell.Second test is when not having linker between GFP and the Vpr albumen, and proteolytic enzyme does not influence the GFP-Vpr melts and arrives the nuclear membrane position, and proteolytic enzyme just can't be sheared and discharges GFP (Fig. 9) like this.Yet when having the classical restriction enzyme site at picture intercellular substance albumen and HIV-1 capsid protein binding site place between GFP and the Vpr albumen, the existence of this proteolytic enzyme can make the location of GFP very big variation (Figure 10) occur.As there not being this proteolytic enzyme, GFP-SL MA-Vpr arrives the nuclear membrane position so, after this proteinase gene is expressed, is sheared and the GFP that discharges will arrive each position of cell, and this moment, most nucleus all can lose mark.Equally, when between GFP and the Vpr albumen if when having the restriction enzyme site of p6 and HIV-1 (Dunn, Goodenow etc., 2002) proteolytic enzyme, the expression of this proteolytic enzyme can make the nuclear membrane of GFP locate the location (Figure 11) of changing whole cell into.This contrast experiment shows that the linker between GFP and the Vpr albumen must exist a restriction enzyme site to reorientate.If there is not known HIV-1 protease cutting site in the linker, and there is an anthrax lethal (Cummings, Salowe etc., 2002) restriction enzyme site, then GFP-SLA-Vpr is conjugated protein all is positioned at nuclear membrane position (Figure 12) before and after proteolytic enzyme is expressed.

GFP location can be used for the sxemiquantitative experiment of enzymic activity, also can be used to have the simultaneous test between two kinds of bacterium colonies of integral protein enzyme gene.When being compared, the growth characteristics of two kinds of bacterium colonies being integrated with proteinase gene finds that the growth-inhibiting phenomenon of first kind of bacterium colony (called after PrIntA or RE294) is more obvious than second kind of bacterium colony (naming PrIntB or RE295).When the nmt1 promotor be suppressed (+when T) back two kinds of colony growths were all acted normally, the PrIntA bacterium colony (T) stopped growth basically, and PrIntB bacterium colony growth velocity in same medium was slowed down in the inducibility medium.The effect that considerable change appears in growth rate has shown that the PrIntA bacterium colony expressed high-caliber proteolytic enzyme, and integrator gene enters chromosomal different modes has also been explained enzyme gene the PrIntA bacterium colony from another angle high expression level phenomenon between two kinds of bacterium colonies simultaneously.The proteinase gene of single copy is incorporated into the nmt1 site, and the difference in point of crossing causes the small segment sequence of nmt1 promotor in two kinds of bacterium colonies difference (Figure 14) to occur.Behind the transfection nmt1 expression vector, the cut and insertion cDNA (Maundrell, 1993) of the NdeI restriction site on the nmt1 promotor.The nmt1 promoter mutation body of this NdeI of not having restriction site can be used for integral protein enzyme gene, and the karyomit(e) that the integration of reorganization homologous gene takes place has the primary type sequence similar to the NdeI agretope.The concrete site that the homologous recombination gene inserts has determined that the proteolytic enzyme of nmt1 promoter expression is primary sequence or mutant sequence.The protease-based of discovering nmt1 promoter expression in the PrIntA bacterium colony is because the primary sequence of NdeI restriction site, and that express in the PrIntB bacterium colony is mutant nucleotide sequence (Figure 14).The adjusting sequence of nmt1 promotor has comprised NdeI restriction site sequence (Zurlinden and Schweingruber, 1997), is exactly high level expression and intensive growth-inhibiting phenomenon that this primary type sequence has caused proteolytic enzyme in the possible PrIntA bacterium colony.

The degree that GFP reorientates among the GFP-SL MA-Vpr has shown that also protease activities is higher than the PrIntB bacterium colony in the PrIntA bacterium colony.When occurring GFP-SL MA-Vpr in the cell, by measuring the distribution situation that fluorescence can react GFP, perhaps be distributed in the whole cell, or according to the Vpr signal distributions in the nuclear membrane surface.Cell is counted as GFP type and Vpr type respectively in mensuration.Some are also arranged between the intermediary cell, they all detect green fluorescence at nuclear membrane and whole cell, and these cells just are counted as the Vpr type.According to this rule, when cell did not have protease activity, nearly all GFP accumulated in the nuclear membrane surface, and promptly GFP profile cell is 0%.When integrator gene was expressed in the PrIntA bacterium colony, about 84% cell was GFP profile (Figure 10-15).And the PrIntB bacterium colony has only 47% cell is the GFP type.The ratio of growth rate in the inducibility medium and GFP type cell has shown that simultaneously the enzymic activity among the PrIntB is lower than the PrIntA bacterium colony like this.

How protein antagonist medicine indinavir influences the PrIntA bacterium colony if having been set forth a protease activity inhibitor, it can be used for the screening of enzyme inhibitor simultaneously.Proteinase inhibitor is the most effective class medicine that treatment HIV infects, and indinavir is one of them medicine.These medicines combine (Randolph and DeGoey, 2004) competitively with enzyme active center.Treat PrIntA with indinavir, then induce the effect that produces proteinase gene to reduce.In-T medium, induce produce proteinase gene after, 12.5 the indinavir of μ g/ml concentration can increase growth velocity, switch to-the T medium in the about 25 hours speeds of growth in back reach maximum value, approach to induce produce proteinase gene+reproduction speed (Figure 16) in the T medium.Enzymic activity that also can reaction suppressor in the green fluorescence distribution phenomenon in the GFP-SL MA-Vpr binding substances suppresses phenomenon.Great majority do not have treated cell to have the distribution of GFP type, and along with the increase of indinavir concentration, (Figure 17) appears in Vpr type cell continually.The distribution pattern of statistics cell, along with the increase of indinavir concentration, the number of GFP type cell reduces (Figure 18) gradually, and when concentration reached the highest 200 μ g/ml, the cell that few part is only arranged was the GFP type.

The experiment of indinavir has shown that directly the PrIntA bacterium colony can be used for the screening of enzyme inhibitors.The basic ideas of screening process are as follows: supressor can make PrIntA form bacterium colony in the induction type substratum, and the proteolytic enzyme toxic action of normal expression can influence the colony that generates normal size.With in 500 cell cultures and induction type and the inhibition type medium, after 5 days, most cell has grown up to colony in the inhibition type medium respectively, and does not almost have in the induction type medium or only formed very little settlement (Figure 19).(two bigger colonies that cause owing to the proteinase gene disappearance in the slide 19 will be introduced in following content).Indinavir joined promptly formed colony in the induction type medium, it is similar to add the colony size that forms in the colony size that forms behind the indinavir of high density and the inhibition type medium substratum.

Illustration 3

Carry out the inhibition shaker test by reducing background values

Set forth the screening that the PrIntA bacterium colony can be used for inhibition before, carry out this test for making in the screening process background be reduced to minimum value.Some these floor cellses in the induction type medium are because lost protease-based thereby grown up to colony after the homologous gene reorganization, and a three-step approach program that finds out recently can be eliminated this floor cells that loses proteinase gene.The operation of this three-step approach is a very classical inhibition screening process, and test has proved that also it can eliminate the influence of background, and this operation comprises 1) will cultivate in cell and the induction type medium; 2) carry out the test of G418 resistance screening after duplicating; 3) check that anti-G418 cell forms the growth of colony situation inducing under the ambient condition.Use this three steps methodology this floor cells that RE294 has carried out research back discovery arrestin enzyme gene is formed the ability of colony less than 1/106.

At first, will cultivate PrIntA cell that the carrier that grows up to carries in inhibition type medium switches in the induction type medium and (T) cultivates.Since the expression of proteinase gene, most necrocytosis, and about 1/1000 cell has grown up to colony (Figure 20).Look as if most of even all colonies all are that to lack cell by the proteinase gene that homologous recombination gene and promotor repeated overlapping cause formed, lose proteinase gene in this way and return the disappearance (Fig. 3) that causes the kan gene.The fission yeast cell that contains the Kan gene can be resisted the G418 microbiotic, induction type matrix is copied to contain G418 matrix substratum and cultivate to identify whether there is the kan gene.Also induction type matrix is transferred to simultaneously and do not contained experiment in contrast in the antibiotic YE medium of G418.In this control experiment, to similar in the initial medium, all colonies all grow up to the colony of same cell type.On the contrary, transfer to colony on the G418 culture plate from induction type matrix, major part has all stopped growth (FIG.21), show that most cell has all lost the kan gene, and the growth of these colonies in inducing medium is caused by the disappearance of proteinase gene mainly.

Yet, approximately have only 1% colony on the G418 copy board growth (wherein two in Figure 21 as seen).Discover that further these colonies in fact are not because the restraining effect of enzyme has stopped growth owing to various cell mixing in inducing medium forms colony.As if after these anti-G418 colonies were tested in the inducing culture plate again, they had all stopped growth, and in control experiment, remain with proteinase gene in these bacterium colonies and this expression of gene is still had susceptibility (Figure 22).Two kinds of contrast bacterium colonies that grow up to from the cell of G418 antibiotic-screening after losing proteinase gene, grow better (on Figure 22), the PrIntA bacterium is in the starting stage of G418 cultivation, the cell that has only sub-fraction to lose proteinase gene has grown up to a few bigger colony (Figure 23 periphery), and most cell stops growing.To cultivate and induce when detecting once more the medium from the growth form colony of G418 antibiotic-screening, most cell be consistent with the initial behavior of PrIntA bacterium in G418, does not promptly grow up to the colony (six parts of all the other of Figure 22) of normal size.

The mixture of G418r albumen and G418s albuminous cell exists simultaneously, is that formation is this after initially inducing the colony adding G418 that grows in the medium to carry out resistance screening, and cell is the major cause of dormant opposition phenomenon.Cell is induced when growing up to colony in the matrix original, and the cell that loses proteinase gene and kanr gene grows up to the colony of no G418 albuminous cell soon.But a part of cell in the colony can still contain G418r albumen.This matrix is copied on the G418 culture plate of rich vitamin B, because VITAMIN can arrestin enzyme expression of gene, the G418r albuminous cell of minority has just grown up to colony, and the proteic cell of no G418 is then by antibiotic kills.Granulocyte colony on the G418 copy board is cultivated with inducing and has promptly been stopped growth in the medium as can be seen in Figure 22.

From the experiment of PrIntA vector selection inhibition, will greater than 106 cell cultures with induce in the medium, these media copied to contain in the G418 substratum, in inducing medium, granulocyte colony is carried out antibiotic-screening once more then.After the screening of having finished these three steps, the colony that the G418 microbiotic is produced resistance promptly be separated (Figure 23).This result shows in the screening process of three steps, reaches hour at background, still can the protein inhibitor of only a few be separated.

Illustration 4

The screening experiment in fission yeast karyomit(e) storehouse

In the process in the screening plasmid storehouse of these classics, have some background colonies to see through this three steps (Figure 20), but these micro-colonies can not produce great influence to the separation and the screening of plasmid inhibition.In addition, losing of plasmid in the screening process increased an experimental procedure can improve the reliability of experiment, but prerequisite needs service area ground to divide the background bacterium colony and has the bacterium colony that target suppresses plasmid.The Sill yeast chromosomal group of using in the experiment of splitting is carried (Barbet, Muriel etc., 1992) by the pUR18 carrier that has the ura4 gene.In the process of the pUR18 vector plasmid being carried out the protease inhibitor screening, each cell has 5-10 plasmid that the expression phenomenon took place; The anti-G418 cell that can grow in-T matrix is the target cell that suppresses the plasmid screening, under some special situations, detects the influence that growing state in-T medium can be discharged the cell of a handful of plasmid loss effectively.

Gene integration in the plasmid in the cytogene storehouse after, on average each cell can copy to 5-10 doubly more than.PrIntA is transfected in gene pool, suppresses to select on the plate about 40000 ura4+ genes to come arrestin enzyme expression of gene at one.The screening of carrying out the 3rd step in the medium is cultivated and induced to cell after transfected again.

We have separated the bacterium colony of two kinds of energy strongly inhibited proteolytic enzyme in to the screening process of transfection body, find that these two kinds of bacterium colonies all contain the mutator gene of this proteolytic enzyme but further analyze the back.These two kinds of bacterium colonies well-grown in containing the medium of G418, and PrIntA the bacterium colony lucky and transfection of pUR18 vector plasmid has formed contrast in inducing medium, the PrIntA bacterium colony that has a transfection plasmid is grown also better in G418 matrix, and then most cell stops growing in inducing medium.The seemingly good pUR18 carrier enzyme of these two kinds of bacterium colonies suppresses the carrier of plasmid, in case behind this plasmid loss, these two kinds of bacterium colonies also can continue growth (Figure 25) in inducing medium.The plasmid that obtains in pUR18 gene pool less stable in fission yeast, can not screen plasmid be rich in the uridylic medium growth after, some cells stop growing after will losing plasmid, and only could continued growth under the condition of adding uridylic.In the uppermost culture plate the single colony of growing in the non-selectivity substratum is detected among Figure 25, under the condition of not adding uridylic, can grow and determine whether to contain plasmid (the pUR18 vector plasmid that has the ura4 gene) by observing this bacterium colony.Above two-part bacterium colony stopped growth and shown that this bacterium colony lost plasmid, on the contrary, all the other keep four parts of growth to show that plasmid do not lose.When placing the inhibition medium that has added uridylic to detect same single bacterium colony (Figure 25 bottom left section), all six colony growth situations are consistent with expection, and in inducing medium, grow too good (lower right-most portion of Figure 25).These experiments have shown that the plasmid in these bacterium colonies does not carry inhibition.We have also found to have second kind of bacterium colony of same nature in this screening process.

We have carried out the pcr amplification order-checking to these the two kinds proteinase genes that do not contain in the inhibition plasmid bacterium colony.All there be the 18th the amino acid transposition jumping phenomenon (Figure 26) behind the codon in proteinase gene in two bacterium colonies, and this sudden change can cause the inactivation of enzyme.Two transpositions sudden change all occurs in the 6G position in the sub-16-17 of original password zone, more than one a guanine, another has then lacked a guanine.Two kinds of transposition sudden changes with the zone have shown that simultaneously 6G is the higher zone of frequency of undergoing mutation.In addition, the frequency of enzyme gene generation spontaneous mutation is exactly minimum background values, can detect in screening process, and after two kinds of spontaneous mutation things were separated, whole screening process had just reached minimum background values.After will mixing at the plasmid that screening process is lost, to such an extent as to background reaches the minimum false positive (introducing) of only finding in following content in whole gene pool screening process.The background values that relates in screening inhibition plasmid process is as follows: during at 3 * 105 cells of screening, only find that two anti-G418 bacterium colonies have very strong energy for growth in-T, this strong energy for growth does not need plasmid, and all there is the reading frame displacement sudden change in the proteinase gene, an only false positive bacterium colony in-T medium energy for growth a little less than, in yeast, detect and find from this false positive bacterium colony, to separate the plasmid that obtains and do not have the inhibition ability.

Illustration five

Typical plasmid supressor carries the HHP2 gene

After finishing by plasmid screening inhibition, we have separated 11 kinds of inhibited bacterium colonies, and Figure 27 has shown 6 weak inhibitor are wherein copied to the multiple screening process (being labeled as PS3 respectively, PS4, PS5, PS6 and PS7) of inducing the medium from G418.These six are suppressed bacterium colony and grow in G418 (top culture dish) and inhibition culture dish (left bottom culture dish) better, and it is slower in induction type culture dish (bottom of right side) growth, be different from general 3-4 days, just long to medium sized colony after cultivating six days.The plasmid loss of PS4 has shown that the inhibition ability of these weak proteinase inhibitor depends primarily on plasmid wherein among Figure 28.Four single colonies that form after PS4 grows in the non-selectivity substratum are labeled as PS4-1 respectively, PS4-2, PS4-3 and PS4-4, plasmid to these four single colonies in selectivity matrix has carried out analyzing (top culture dish), wherein PS4-1 and PS4-2 have lost plasmid, and all the other two are still keeping plasmid.These four colonies all are grown in the left bottom position of inhibition culture dish, and the PS4-3 and the PS4-4 that wherein have plasmid have grown up to many colonies in inducing medium, and major part has not stopped growth with the PS4-1 and the PS4-2 cell of plasmid.

In order to have supressor in the plasmid of confirming these 11 kinds of bacterium colonies, utilize these bacterium colonies to prepare the transfection that DNA is used for E.coli, plasmid is reclaimed in amicillin resistance screening back.The supressor of carrying on the plasmid through last confirm after, the plasmid that reclaims transfection is again redeterminated its inhibition ability then to PrIntA.In E.coli, reclaim pPS3-2 and pPS4-10 plasmid in PS2 and the PS3 two primary yeasts inhibition bacterial strain, detect two kinds of PrIntA transfection bodies.The pPS3-2 plasmid is not a protease inhibitor, because the growth phenomenon of two transfection bodies consistent with the pUR18 carrier (Figure 29).PPS3 is a unique bacterium colony that has plasmid that screens steps by all, but plasmid is wherein reclaimed also transfection again behind PrIntA, and the tool inhibition is active after the detection.For the pPS4-10 plasmid, two transfection bodies in inducing medium upgrowth situation all than pUR18 carrier and pPS3-2 plasmid transfection body good (Figure 29).Show that the pPS4-10 plasmid has more weak active supressor.Nine plasmids of other that reclaim from weak inhibition yeast colony also have certain growth phenomenon (Figure 30) in inducing medium.Measure its plating efficiency when pPS3-2 plasmid and pUR18 carrier are imported back 24 hours and have only about 0.4%, and the plating efficiency Danone of the plasmid transfection body of other ten recovery reaches 7% (Figure 31).

600 Nucleotide that insert the gene end in these ten inhibition plasmids are checked order, find that they only carry hhp2 gene (Figure 32).Ten plasmids all have complete hhp2 opening code-reading frame (ORF), and the gene at contiguous position does not all have ORF.These ten hhp2 plasmids that obtain in 40000 transfection bodies that from gene pool, screen, we have separated six different plasmids, and they come from different transfection bodies.Separating for several times all is because derive from same transfection pond or same transfection body has been carried out repeated isolation to three kinds of situations of same plasmid.All weak inhibition plasmids all comprise the hhp2 gene: suppress institute that bacterium colony passed through the plasmid loss screening a little less than in the of ten in steps, with separate in these bacterium colonies the plasmid obtain again transfection detect behind the fission yeast and find that more weak inhibition activity is all arranged, they only have the hhp2 gene; These ten plasmids have comprised six different hhp2 plasmids.

Because hhp2 separates to obtain from six different transfection ponds, and does not detect other gene, infer the hhp2 gene be in the pUR18 gene pool a unique energy arrestin enzymic activity at the screening goal gene.We do not find the hhp1 gene in the enzyme inhibition factor screening process.Hhp1 and hhp2 gene order closely similar (74% is consistent), and have the function of overlapping (Dhillon and Hoekstra, 1994).

The evaluation of hhp2 gene got rid of to have suppressed active possibility.The high level expression of enzyme can be to the yeast toxigenicity, because an indispensable protein in the yeast is decomposed after digestion is low to moderate finite concentration by this kind of enzyme, cell will lose viability.In this model,, just can reduce toxic side effect if cell can synthesize abundant indispensable protein.But this model is not to be used for hhp2, because the bacterium colony of hhp2 genetically deficient still has good viability (Dhillon and Hoekstra, 1994).Other suppressor gene can not be carried out isolating reason may be that this proteolytic enzyme acts on a plurality of necessary albumen simultaneously, or has other toxic mechanism in the alternative process.

The concrete mechanism of hhp2 arrestin enzyme also will further be studied.The hhp2 kinases belongs to CK1 family kinases (Dhillon and Hoekstra 1994), does not at present have relevant report for this class kinases with interaction mechanism between the HIV-1 proteolytic enzyme.We have introduced the mechanism of such hhp2 activating enzyme arrestin expression of enzymes in this patent.First kind is the expression that hhp2 passes through phosphorylation approach arrestin enzyme.In the concrete process of another kind, this kinases is not with the proteolytic enzyme phosphorylation, and hhp2 is decomposed by proteolytic enzyme as a kind of emulative antagonist.We have confirmed whether hhp2 suppresses HIV-1 proteolytic enzyme by a kind of new approach in this patent of invention.

Confirmation for hhp2 has shown that directly the screening process that we invented is the process of a very effective screening supressor.Hhp2 is one of them gene (Wood, Gwilliam etc., 2002) in 5000 genes of fission yeast, and our screening process can find this gene from six different plasmids.The screening process that shows this classics can be used to express the screening of protease inhibitor in the gene pool of greater protein matter and peptide compounds.

Reference

Below listed all patents, patent application and the documents and materials published shown the knowledge and skills level that person skilled had in field of the present invention.These patents, patent application and the documents and materials of publishing are incorporated herein this paper as a reference.

Patent and patent application

Application No. 5,384,253

Application No. 5,399,363

Application No. 5,466,468

Application No. 5,543,158

Application No. 5,580,579

Application No. 5,629,001

Application No. 5,641,515

Application No. 5,725,871

Application No. 5,756,353

Application No. 5,780,045

Application No. 5,804,212

Application No. 5,792,451

Application No. 6,043,357

Application No. 6,613,308

Zhu Ce invention H1 in accordance with the law, 649

The documents and materials of publishing

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Chen，M.，R.T.Elder，et al(1999).“Mutational analysis of Vpr-inducedG2arrest，nuclear localization，and cell death in fission yease.”J Virol 73(4)：3236-45.

Cummings，R.T.，S.P.Salowe，et al.(2002).“A peptide-based fluorescenceresonance resonance energy transfer assay for Bacillus anthracis lethalfactor protease.”Proc Natl Acad Sci USA 99(10)：6603-6.

Dhillon，N.and M.F.Hoekstra(1994).“Characterization of two proteinkinases from Schizosaccharomyces pombe involved in the regulation of DNArepair.”Embo J 13(12)：2777-88.

Dunn，B.M.，M.M.Goodenow，et al.(2002).“Retroviral proteases.”Genome Biol 3(4)：REVIEWS3006.

Keeney，J.B.and J.D.Boeke(1994).“Efficient targeted integration atleul-32 and ura 4-294 in Schizosaccharomyces pombe.”Genetics 136(3)：849-56.

Maundrell，K.(1993).“-Thiamine-repressible expression vectors pREPand pRIP for fission yeast.”Gene 123(1)：127-30.

Randolph，J.T.and D.A.DeGoey(2004).“Peptidomimetic inhibitors ofHIV protease.”Curr Top Med Chem 4(10)：1079-95.

Remington’s Pharmaceutical Sciences，18 ^th Ed.Mack Printing Company，1990.

Tommasino，M.and K.Maundrell(1991).“Uptake of thiamine bySchizosaccharomyces pombe and its effect as a transcriptional regulator ofthiamine-sensitive genes.”Curr Genet 20(1-2)：63-6.

Wlodawer，A.and A.Gustchina(2000).“Structural and biochemicalstudies of retroviral proteasesl”Biochim Biophys Acta 1477(1)：16-34.

Wood，V.，R.Gwilliam，etal.(2002).“The genome sequence ofSchizosaccharomyces pombe.”Nature 415(6874)：871-80.

Zurlinden，A.and M.E.Schweingruber(1997).“Identification of a DNAelement in the fission yeast Schizosaccharomyces pombe nmt1(thi3)promoter involved in thiamine-regulated gene expression.”J Bacteriol179(18)：5956-8.

Though above described technical scheme of the present invention and technical progress in detail, those skilled in the art can make some changes, replacement, change on the basis that does not break away from invention spirit of the present invention and the determined protection domain of claims of the present invention.In addition, given practical range is not limited to the embodiment of process listed in the specification sheets, equipment, structure, material composition, method, step.That existed from the disclosed content of the present patent application file, prior art and subsequently with technology such as the process, equipment, structure, material composition, method, step of development, any one those skilled in the art can realize with the corresponding embodiment identical functions of the present invention fully or reach identical technique effect.Therefore, the effect of claims is the protection domain of the definite process that will protect, equipment, structure, material composition, method, step.

Claims (44)

1, a kind of method of differentiating the hiv protease inhibitor, this method may further comprise the steps:

A kind of yeast cell at first is provided, includes the polynucleotide of forming by the gene order of coding hiv protease in the described yeast cell;

Then described polynucleotide is inserted in the candidate agent;

Reevaluate following wherein one or two indexs:

1) cytoactive of described yeast cell and/or growing state; Wherein, compare with cell under the situation that lacks candidate agent, cell can be survived under the situation that described candidate agent exists, and described candidate agent is the hiv protease inhibitor;

2) enzyme of hiv protease substrate is cut effect, and wherein can stop the digested candidate agent of substrate is the hiv protease inhibitor.

2, method according to claim 1 is characterized in that: described hiv protease is HIV-1 proteolytic enzyme.

3, method according to claim 1 is characterized in that: described hiv protease is HIV-2 proteolytic enzyme.

4, method according to claim 1 is characterized in that: the adjusting of expression regulation sequence has been in the expression of described hiv protease.

5, method according to claim 1 is characterized in that: described inhibitor is polypeptide, polynucleotide, small molecules, antibody, perhaps the mixture or the composition of above multiple material.

6, method according to claim 1 is characterized in that: described yeast is a fission yeast.

7, method according to claim 6 is characterized in that: described fission yeast is a schizosaccharomyces pombe.

8, method according to claim 1 is characterized in that: described polynucleotide is integrated in the yeast genes group.

9, method according to claim 4 is characterized in that: described expression regulation sequence excessively is that nml1 crosses expression regulation sequence, adh1 regulating and controlling sequence, fbp1 regulating and controlling sequence, inv1 regulating and controlling sequence or ctr4 regulating and controlling sequence.

10, method according to claim 1 is characterized in that: include at least one can reduce the background growth signals in the cytoactive test fragment in the described polynucleotide.

11, method according to claim 10 is characterized in that: the described fragment that can reduce the background growth signals is a marker.

12, method according to claim 11 is characterized in that: the marker of described marker for microbiotic is had resistance.

13, method according to claim 11 is characterized in that: the marker of described marker for G418 or Totomycin are had resistance.

14, method according to claim 11 is characterized in that: described marker is a nutritional marker.

15, method according to claim 11 is characterized in that: described marker is selected from ade1, ade6, arg3, CAN1, his3, his7, leu1, leu2, sup3-5, ura3 and ura3.

16, method according to claim 1 is characterized in that: described hiv protease behaviour hiv protease.

17, method according to claim 1 is characterized in that: described hiv protease substrate behaviour hiv protease substrate.

18, method according to claim 1, it is characterized in that: the enzyme of assessment hiv protease substrate is cut effect can cut effect for the enzyme that assessment is formed polypeptide by first polypeptide fragment and second polypeptide fragment, and wherein the restriction enzyme site of hiv protease is between described first polypeptide fragment and second polypeptide fragment.

19, method according to claim 18 is characterized in that: be detectable mark in first polypeptide fragment.

20, method according to claim 18 is characterized in that: second polypeptide fragment is the substrate of hiv protease.

21, method according to claim 20 is characterized in that: the substrate of described hiv protease is the HIV-1Vpr polypeptide.

22, method according to claim 1 is characterized in that: the restriction enzyme site of described hiv protease substrate can be DSQNYPIVQ (SEQ ID NO:3), DSFNSTQIT (SEQ ID NO:4), VSQNYPIVQN (SEQ ID NO:8), KARVLAEAMS (SEQ ID NO:9), SATIMMQRGN (SEQ ID NO:10), RPGNFLQSRP (SEQ ID NO:11), VSPNFPQITL (SEQ ID NO:12), CTLNFPISPI (SEQ ID NO:13), GAETFYVDG (SEQ ID NO:14) or IRKVLFLDGI (SEQ ID NO:15).

23, method according to claim 19 is characterized in that: described detectable green fluorescent protein, emerald green fluorescin, yellow fluorescence protein, blue fluorescent protein or the cyan fluorescent protein of being labeled as.

24, method according to claim 1 is characterized in that: this method comprises further that also the described inhibitor with the treatment effective dose is transported to the step of carrying HIV or suffering from the individuality of AIDS.

25, method according to claim 1 is characterized in that: this method comprises further that also the described inhibitor with effective dose is transported to PI HIV virus and maybe may suffers from the step that the individuality of AIDS is used as preventing.

26, treat the method that has infected HIV virus or suffered from the individuality of AIDS, this method comprises that the pharmaceutical composition that includes hhp2 that will treat effective dose is transported to the step of described individuality.

27, method according to claim 26 is characterized in that: described hhp2 is a polypeptide.

28, method according to claim 26 is characterized in that: described hhp2 is the polynucleotide of coding hhp2 polypeptide.

29, a kind of yeast cell is characterized in that: include the polynucleotide of being made up of the gene order of coding hiv protease in this yeast cell.

30, yeast cell according to claim 29 is characterized in that: described hiv protease is a HIV-1 proteolytic enzyme.

31, yeast cell according to claim 29 is characterized in that: described hiv protease is a HIV-2 proteolytic enzyme.

32, yeast cell according to claim 29 is characterized in that: the adjusting of expression regulation sequence has been in the expression of described polynucleotide.

33, yeast cell according to claim 29 is characterized in that: the preferred marker of described polynucleotide.

34, yeast cell according to claim 33 is characterized in that: described marker preferably has the marker of resistance to microbiotic.

35, yeast cell according to claim 33 is characterized in that: the preferred nutritional marker of described marker.

36, yeast cell according to claim 29, it is characterized in that: include the polynucleotide of being made up of second sequence of first sequence of coding first polypeptide fragment and second polypeptide fragment of encoding in the described yeast cell, wherein the restriction enzyme site of hiv protease is between first polypeptide fragment and second polypeptide fragment.

37, yeast cell according to claim 36 is characterized in that: described first polypeptide fragment is detectable mark.

38, according to the described yeast cell of claim 37, it is characterized in that: described detectable green fluorescent protein, emerald green fluorescin, yellow fluorescence protein, blue fluorescent protein or the cyan fluorescent protein of being labeled as.

39, yeast cell according to claim 36 is characterized in that: described second polypeptide fragment is the HIV-1Vpr polypeptide.

40, yeast cell according to claim 29 is characterized in that: described yeast cell can be yeast cell culture or yeast cell colony.

41, a kind of pharmaceutical composition test kit that treats and/or prevents infected by HIV and/or suffer from AIDS is characterized in that: this test kit includes polynucleotide or the hhp2 polypeptide of the coding hhp2 that is contained in the suitable containers.

42,, it is characterized in that this test kit also includes pharmaceutically acceptable excipient according to the described test kit of claim 41.

43, a kind of test kit that is used to screen one or more hiv protease inhibitor, this test kit includes the yeast cell described in the claim 29.

44, according to the described test kit of claim 43, it is characterized in that: also include the polynucleotide of being made up of second sequence of first sequence of coding first polypeptide fragment and second polypeptide fragment of encoding in the described yeast cell, wherein the restriction enzyme site of hiv protease is between first polypeptide fragment and second polypeptide fragment.

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