CN101671653B - Screening system of HIV infected cell and applications thereof - Google Patents

Abstract

The invention provides a screening system of HIV infected cell. A reporter protein gene is divided into a reporter gene N-end and a reporter gene C-end, wherein the reporter gene N-end and dnae-N which has the protein splicing function and is the part of DnaE intein N-end gene are blended to form an expression vector I; and the reporter gene C-end and dnae-C which has the protein splicing function and is the part of DnaE intein C-end gene are blended to form an expression vector II. The two expression vectors respectively transfect an HEK293 cell or a CHO cell containing chemotactic factors and CD4 protein or containing envelope protein Env of HIV, and establish stable expression cell stains. When the two strains of stable cells are cultured mixedly, the cells are blended, thus promoting DnaE-C and DnaE-N to mutually contact and act; while the combined action of DnaE-N and DnaE-C connects the reporter protein N-end and the reporter protein C-end into one complete reporter protein, and finally, the blending degree can be learned by the protein activity of a test report.

Description

A kind of screening system of HIV infected cell and application thereof

(1) technical field

The present invention relates to a kind of medicament sifting motion system of cell-cytogamy of the HIV of simulation infected cell, and the application in screening chemokine ccr 5 and CXCR4 antagonist.

(2) background technology

The envelope glycoprotein gene env of HIV from being transported to the tenuigenin by in the nucleus that infects, synthesizes precursor protein gp160 then on rough endoplasmic reticulum under the proteic effect of Rev after being encoded into mRNA; The gp160 guiding of the signal peptide on the gp41 of the portion territory within it gets in the endoplasmic down immediately, the formation of concurrent living intramolecular disulfide bond, oligomerization, gp120 territory part height glycosylation modified; Transferring to from endoplasmic reticulum the process of golgi body, the gp160 precursor protein cuts under the effect of host protein enzyme (furinora furin-like enzyme), forms sophisticated envelope glycoprotein gp120 and transmembrane glycoprotein gp41 complex body.The envelope glycoprotein complex body is anchored on the cytolemma through gp41 with trimerical form, is connected through non covalent bond between gp41 and the gp120.

The first step of HIV infected cell is that gp120 identification is by people's differentiation antigen 4 PROTEIN C D4 of infected cell.Behind the CD4 albumen in conjunction with cell; The conformation of gp120 changes; Cause auxiliary receptor CCR 5 or CXCR4 further to combine above-mentioned complex body, make gp41 that further conformational change take place, cause HIV coating that gp41 participates in by infected cell combine finally cause the film fusion.

Traditional screening CCR5 antagonist method is I ¹²⁵The part of mark combines method of testing.But nearest discovers, part combines method of testing to obtain the CCR5 potential antagonist or antibody becomes irrelevance in the result that the antivirus action analytical procedure obtains.Because HIV virus is infectious to experimental implementation person's potential, screens HIV on a large scale through antiviral infestation method and infect suppressor factor and seem unrealistic.

The alternative detection method of having reported multiple simulation HIV virus-cytogamy has been arranged at present.Can be divided into two big types substantially: false HIV virus-cytogamy method of testing and cell-cytogamy method.False HIV virus-cytogamy method of testing is as fusion vector through viral perhaps other virus particle that has the HIV envelope glycoprotein of the HIV of reduction.The defective of this method is that each analysis needs to prepare new virus particle, and expends very long incubation time.This makes and is difficult to apply to extensive shaker test.Cell-cytogamy method of testing can merge with the cell that does not infect after by the HIV virus infection based on the human cell, and the mechanism of this amalgamation mode is similar and the mode of virus infection cell.Utilization different cells system and fusion detection method; Many testing method have been arranged at present; This comprising: 1, dye observation of cell-intercellular fusion through cytolemma and nucleus, the operation steps that this method is tediously long is not suitable for the large-scale medicine shaker test.2, detect the fusion of cell through the expression of transcriptional activation signal gene after the cytogamy, this method has applied to extensive shaker test, but owing to need expend certain hour through the signal protein transcriptional activation, so screening time has much room for improvement.

Intein (intein) is from one section aminoacid sequence in protein translation product (precursor protein) reading frame of bacterium; The mode that it leans on the oneself to shear discharges from precursor protein, and with peptide bond the two ends peptide chain being linked to each other simultaneously forms ripe proteic mode mediating protein montage (splicing).The DnaE intein from blue-green algae Synechocystis sp.PCC6803 of report in 1998 is a kind of intein; The gene of its N end and C end montage structural domain is separated by the genome sequence of 750kb, and the protein translation product still can be accomplished extein (DnaE through the intersegmental identification of intein sheet, reconstruction _Ext-N and DnaE _Ext-C) montage finally forms complete DanE _ExtAlbumen.The DnaE intein is present in in addition a little blue-green algaes equally.The montage characteristic of DnaE intein is applied to albumen cyclisation, protein purification, protein fragments isotopic labeling etc., particularly is applied to interaction between protein.Have 2 montage fragments that interaction protein is integrated into DnaE intein respectively with 2, directly hold with N again to link to each other with the C end of reporter protein (but like the luciferase of luminous detection, enhanced green fluorescence protein etc.).Two protein-interactings cause intein each other near so that the folding montage activity that demonstrates; Make protein-active that two non-activity reporter protein fragments of expressing at different carriers respectively regain one's integrity and luminous, detect and the interphase interaction of quantitative and qualitative albumen through light.Reported that monitoring plastosome with this method discharges albumen, nuclear locating sequence etc.

(3) summary of the invention

The oneself that the present invention uses bacterium DnaE intein shears the characteristics that function and HIV virus envelope protein Env and human CD4 albumen and CCR5 or the interphase interaction of CXCR4 chemokine protein can inducing cell be merged, and the medicaments sifting model of chemokine mediated cytogamy detection model of a kind of CCR5 of can be used for or CXCR4 and antagonism HIV infected cell is provided.

The technical scheme that the present invention adopts is:

A kind of screening system of HIV infected cell; Comprise Chemokine Receptors screening clone and HIV envelope protein Env screening clone; Said screening system is made up by following method: (1) meets (dnae-C-reptort-C) with the C end of DnaE intein gene (clone's the DnaE intein with albumen splicing function all is suitable for arbitrarily) and the C end matching of reporter gene, inserts and obtains fusion expression vector 1 in the plasmid 1; The N end of DnaE intein gene and the N end matching of reporter gene are met (reptort-N-dnae-N), insert and obtain fusion expression vector 2 in the plasmid 1; (2) human CD 4 protein gene cd4 is inserted plasmid 2, obtain recombinant expression vector 3; Human chemokine CCR5 gene ccr5 or CXCR4 gene cxcr4 are inserted plasmid 1, obtain recombinant expression vector 4; (3) HIV-1 is different virus strain Env gene HXB2-env or SF162-gp160 genes insert plasmid 3, obtain recombinant expression vector 5; (4) with one in carrier 1 or the carrier 2, with carrier 3 and 4 cotransfections to HEK293 cell or Chinese hamster ovary celI, obtain the Chemokine Receptors screening clone of stably express; (5) with in carrier 1 or the carrier 2 another, (step (4) and step (5) are generally used different cells, occur false positive when causing the screening antagonist to prevent using same cell to cause and self merge with carrier 5 cotransfections to Chinese hamster ovary celI or HEK293 cell.If but carry out contrast simultaneously, also be feasible (equaling to have increased a step)), the HIV envelope protein Env that obtains stably express screens clone; Said plasmid 1, plasmid 2, plasmid 3 efficiently express proteic carrier for conventional in mammalian cell, plasmid 1, plasmid 2 can be selected pcDNA3.1 (Invitrogen Company products) for use, pT ARGET ^TM(Promega Company products) etc., two kinds of carriers can be the same or different, and plasmid 3 can be selected pSV7d or pCAGGS for use; Said reporter gene is conventional activated enzyme gene that is used to detect or protein gene that can be luminous; Comprise various luminescent proteins (GFP for example, YFP, CFP; BFP; RFP etc.) and two mutants, also comprise aequorin or clindamycin, and various luciferase, beta-galactosidase enzymes, tyrosine oxidase and other many kinds of enzymes etc.Be divided into two reporter gene; Be reporter gene N end and reporter gene C end; This two part all loses the activity or the fluorescence of enzyme, has only through intein (intern) to be spliced into complete albumen to reporter protein N end and reporter protein C end again, just recovers original enzymic activity or fluorescence.When Chemokine Receptors screened the recombinant vectors of expression of cell lines dnae-C-reptort-C, HIV envelope protein Env screening clone selected to contain the recombinant vectors of reptort-N-dnae-N; Vice versa.

Concrete; Said screening system is made up by following method: (1) (nucleotide sequence is seen SEQ ID NO.1 with DnaE intein gene; Aminoacid sequence is seen SEQ ID NO.2) the C end matching of C end and reporter gene connect, insert and obtain fusion expression vector 1 among the plasmid pcDNA3.1; The N end of DnaE intein gene and the N end matching of reporter gene are connect, insert and obtain fusion expression vector 2 among the plasmid pcDNA3.1; (2) people CD4 gene (nucleotide sequence is seen SEQ ID NO.15, and aminoacid sequence is seen SEQ ID NO.16) is inserted plasmid pFLAG-CMV ^TM-3, obtain recombinant expression vector 3; (nucleotide sequence is seen SEQ ID NO.11 with human chemokine CCR5; Aminoacid sequence is seen SEQ ID NO.12) or CXCR4 (nucleotide sequence is seen SEQ ID NO.13; Aminoacid sequence is seen SEQ ID NO.14) gene insertion plasmid pcDNA3.1, obtain recombinant expression vector 4; (3) (nucleotide sequence is seen SEQ ID NO.19 to get expression HXB2-env; Aminoacid sequence is seen SEQ IDNO.20) or SF162-gp160 (nucleotide sequence is seen SEQ ID NO.17; Aminoacid sequence is seen SEQ ID NO.18) recombinant expression vector 5 of gene; Said carrier 5 can make up voluntarily, also can be provided by NIH AIDS Research and Reference Reagent Program; (4) with one in carrier 1 or the carrier 2, with carrier 3 and 4 cotransfections to HEK293 cell or Chinese hamster ovary celI, obtain the Chemokine Receptors screening clone of stably express; (5) with carrier 1 or carrier 2 another,, obtain the HIV envelope protein Env screening clone of stably express with carrier 5 cotransfections to Chinese hamster ovary celI or HEK293 cell.

When the goal gene behaviour chemokine gene ccr5 that inserts in step (2) recombinant expression vector 4, the goal gene that inserts in step (3) recombinant expression vector 5 is the envelope protein gene SF162 gp160 of HIV-1 virus strain SF162.

When the goal gene behaviour chemokine gene cxcr4 that inserts in step (2) recombinant expression vector 4, the goal gene that inserts in step (3) recombinant expression vector 5 is the envelope protein gene HXB2-env of HIV-1 virus strain HXB2.

Preferably, said reporter gene is one of following: 1. green fluorescent protein (EGFP) gene, 2. red fluorescent protein (DsRed) gene, 3. Renilla luciferase (Renilla luciferase) gene, 4. Firefly luciferase (Firefly luciferase) gene, 5. SEAP (SEAP) gene, 6. Bete-lactamase (Beta-lactamase) gene, the 7. tilactase (gene of β-Gal).

Preferably, said DnaE intein gene dane reads strain algae Nostoc punctiforme PCC73102 from basketball algae Anacystis nidulansR2 PCC7942 or point-like.Concrete DnaE intein gene is read the N end (dnae-N) and the C end (dnae-C) of cloning DnaE intein gene the strain algae Nostoc punctiforme PCC73102 genomic dna from basketball phycomycete strain Anacystis nidulans R2 (PCC7942) or point-like.

The cell levels high-throughput screening of cell lines that the present invention makes up mainly can interact with the HIV envelope glycoprotein Env of cell inner expression based on the DnaE intein of tool albumen self-splicing function and the CCR5/CXCR4 albumen and the CD4 albumen of heterogenous expression; Cytogamy during simulation HIV infected cell, its principle is referring to Fig. 1.By reporter protein such as E.C. 2.3.1.28 (CAT), luciferase (Luc), alkaline phosphatase enzyme (SEAP), tilactase (β-Gal) be divided into two into reporter gene N end and reporter gene C end, reporter gene N end and dnae-N (DnaE intein gene N end) amalgamation and expression with protein splice function with green fluorescent protein (GFP); Reporter gene C end then with the dnae-C with protein splice function (DnaE intein gene C end) amalgamation and expression.With two expression vectors respectively transfection contain HEK293 cell or the Chinese hamster ovary celI of chemokine or contain HEK293 cell or the Chinese hamster ovary celI of HIV envelope protein Env, and be built into stable expression cell strain.When two strain stabilized cell mixed culture; Cell merges; Thereby impel DnaE-C and DnaE-N can be in contact with one another effect; And connect into a complete reporter protein to reporter protein N end and reporter protein C end by DnaE-N and DnaE-C acting in conjunction, just can know the fusion degree of cell at last through the examining report protein-active.

The screening clone that the present invention makes up is to utilize the principle that HIV virus envelope protein Env and human differentiation antigen CD4 albumen and CCR5 or the interphase interaction of CXCR4 chemokine protein can the inducing cell fusion; Irrelevant with the clone source of being selected for use, do not receive the influence of reporter gene type.

In Chemokine Receptors screening clone, chemokine and CD4 albumen overexpression on cytolemma; The fusion fragment of reporter gene and DnaE intein then efficiently expresses in tenuigenin, and reporter gene has no activity.In the HIV envelope glycoprotein Env genescreen clone, the env gene through cutting, forms gp gp120 and gp41, and is anchored on the cytolemma on gorky after transcription and translation becomes precursor protein gp160.The fusion fragment of reporter protein and DnaE then efficiently expresses in tenuigenin, but any activity of tool not.When Chemokine Receptors screening clone and HIV envelope glycoprotein Env genescreen clone mixed culture; Gp120 and CD4 albumen and chemokine interact; The change of occurred conformation impels gp41 to act on Chemokine Receptors screening clone, causes cytogamy.After the cytogamy, sufficient random collision takes place in reporter protein and two complementary segments that merge of DnaE of laying respectively between different clones, DnaE intein generation self-splicing, and, make reporter protein that the activity that regains one's integrity take place to splice.Through the relevant detection instrument, just can detect the activation degree of reporter protein, thereby be used for confirming the integration percentage of cell.

The invention still further relates to the application of described screening system in high flux screening CCR5 or CXCR4 antagonist.

Concrete; Said being applied as: Chemokine Receptors is screened cell line cell and HIV envelope protein Env screening cell line cell add to add and remain the routine of screening of medicaments and be applicable in the substratum (like 1: 1 substratum of DMEM substratum, DMEM/F12, IMDM substratum etc.) of Chinese hamster ovary celI or HEK293 cell; Mixed culture 4～8 hours; Cultured products is through washing, lysis; Adding and the corresponding reaction substrate of reporter protein (if Renilla luciferase reporter protein, with the Renilla luciferase assay systerm of promega company; Reaction substrate is coelenterazine and verivate thereof; If firefly luciferase reporter gene, usefulness be D-luciferin), obtain pharmaceutical activity data to be measured.As reporter gene be can be luminous protein gene, then only need fluorescence intensity, can obtain to survey the pharmaceutical activity data.

As passing through promotor, the reporter gene screening model that trans-acting transcription factor (Tat, T 7 polymerase) makes up; Will be after cell merges through a series of signal transduction; Just can cause reporter gene expression, thus the time number that need hatch hour, long wanted 8 hours.And the screening model that the present invention will make up is a self-splicing function of utilizing DnaE intein; Expressed proteins segment is connected into the reporter protein of complete function, so the activity that just can detect reporter protein in several minutes to half a hour that merges takes place at cell.

The intein that tool is spliced function makes not to be had active reporter gene two portions to close and is that activated enzyme or luminescent protein are a kind of methods of efficient, flexible in order to the mechanism of action between the research intracellular protein; The present invention is applied to detect the cell-intercellular interaction that utilizes HIV membranin and human cell surface protein to set up with this method of use, with making it to become the research instrument of finding or screen prevention and treatment AIDS-treating medicine.This method is the interaction between detection molecules directly, and needn't pass through secondary reaction, like transcriptional activity; The unnecessary macromole labelled protein of using is like complete GFP; More do not need pair cell to dye.This method can be used multiple reporter protein, can be any instrument platform, automatic operation system, cell type and the testing program particular design that needs.According to the selection of reporter protein, can make up the high throughput testing system.In the high throughput testing system, can pass through standard fluorescence microplate reader detection by quantitative fluidic cell, porous plate or microtiter plate.This method can high flux screening and the HIV medicine of the effect of Chemokine Receptors; Also can high flux screening can with the gp120 of the Env genes encoding of HIV or the medicine of gp41 effect, whether final decision has the suitable medicine can antagonism cell-cytogamy effect.

Beneficial effect of the present invention is mainly reflected in: a kind of cell levels high throughput screening system is provided, detected weak point consuming time, and easy and simple to handle.

(4) description of drawings

Fig. 1 is modeling principle figure of the present invention;

The carrier structure synoptic diagram of Fig. 2 for making up among the embodiment; A: recombinant expression vector pCDNA-ccr5; B: recombinant vectors pCMV-flag-cd4; C: plasmid pCAGGS-SF162-gp160; D: fusion expression vector pCDNA-rluc-N-dnae-N; E: fusion expression vector pCDNA-dnae-C-rluc-C;

Fig. 3 screens the fluorescent value synoptic diagram of a collection of CCR5 antagonist gained for utilization the present invention among the embodiment; M: adding concentration is the maraviroc of 30nM, TAK: add the TAK-779 of 1 μ M, T-20: add the T-20 of 1 μ g/ml, ck: positive control;

Fig. 4 is that the CCR5 antagonist maraviroc of different concns is to the restraining effect by the CCR5 cell fusion mediated.

(5) embodiment

Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1:

(1) the N end (dnae-N) and the C end (dnae-C) of clone DnaE intein gene from basketball phycomycete strain Anacystis nidulans R2 (PCC7942) genomic dna; Clone's CCR5 gene (nucleotide sequence is seen SEQ ID NO.11, and aminoacid sequence is seen SEQ ID NO.12) from the human macrophage genome, CD4 gene (nucleotide sequence is seen SEQ IDNO.15, and aminoacid sequence is seen SEQ ID NO.16);

(2) with Anacystis nidulans R2 dnae-C (1-36aa; Nucleotide sequence is seen SEQ IDNO.9; Aminoacid sequence is seen SEQ ID NO.10) (rluc-C, 111-311aa, nucleotide sequence see SEQ ID NO.5 with Renilla luciferase gene C end; Aminoacid sequence is seen SEQ ID NO.6) through overlap PCR dnae-C and rluc-C are stitched together, between dnae-C and rluc-C, add 5 aminoacid sequences of CFNGT; With Renilla luciferase gene N end (rluc-N; 1-110aa; Nucleotide sequence is seen SEQ ID NO.3, and aminoacid sequence is seen SEQ ID NO.4) (37-152aa, nucleotide sequence see SEQ ID NO.7 with Anacystis nidulans R2 dnae-N; Aminoacid sequence is seen SEQ IDNO.8) through overlap PCR rluc-N and dnae-N are stitched together, between rluc-N and dnae-N, add two aminoacid sequences of GS;

(3) with dnae-C and rluc-C and the rluc-N and the dnae-N of above-mentioned structure; Chemokine Receptors gene ccr5 is inserted into respectively among the plasmid pcDNA3.1; Just can obtain fusion expression vector I:pCDNA-dnae-C-rluc-C (Fig. 2-E) and fusion expression vector II:pCDNA-rluc-N-dnae-N (Fig. 2-D), recombinant expression vector pCDNA-ccr5 (Fig. 2-A); The CD4 gene is inserted into expression vector pCMV-flag (pFLAG-CMV ^TMAcquisition recombinant vectors pCMV-flag-cd4-3EXPRESSION VECTOR sigma Company products) (Fig. 2-B); Freely obtain plasmid pCAGGS-SF162-gp160 (Fig. 2-C) from NIH AIDS Research and ReferenceReagent Program;

(4) the HEK293 cell is with 3 * 10 ⁵Density goes down to posterity in 6 porocyte culture plates, in containing the DMEM substratum of 10% foetal calf serum, and 37 ℃, 5%CO ₂Environment is cultivated.Second day with calcium phosphate transfection reagent (invitrogen Company products) cotransfection plasmid pCDNA-dnae-C-rluc-C, pCDNA-ccr5, and pCMV-flag-cd4, the transfection continued was cultivated 12 hours.Chinese hamster ovary celI is with 3 * 10 ⁵Density goes down to posterity in 6 porocyte culture plates, in containing the DMEM/F12 substratum of 10% foetal calf serum, and 37 ℃, 5%CO ₂Environment is cultivated.Second day with Lipofectamine 2000 (invitrogen company) cotransfection plasmid pCAGGS-SF162-gp160, pCDNA-rluc-N-dnae-N, and the transfection continued was cultivated 12 hours.

(5) with the HEK293 cell after the transfection and Chinese hamster ovary celI mixed culture 4 hours in 96 orifice plates, add cytogamy agonist drug to be screened in the substratum before mixing (needing concrete).

(6) substratum in the above-mentioned culture plate is removed in suction; Wash twice with PBS; Use Renilla LuciferaseAssay System (Promega Company products) lysate lysing cell again; Add reaction substrate (coelenterazine) by the test kit requirement, measure fluorescence intensity (time-delay 1s detects 2s) with chemiluminescence detector FB12luminometer (Berthold Company products); The result is as shown in Figure 3; By knowing among the figure: concentration be 30nM maraviroc (the CAS accession number: 376348-65-1), the TAK-779 of 1 μ M (CAS accession number: 229005-80-5) with T-20 (trade(brand)name: Fuzeon, the chemical name: entuvirtide) the CCR5 cell fusion mediated is all had the obvious suppression effect of 1 μ g/ml.The CCR5 antagonist maraviroc of different concns sees Fig. 4 to the restraining effect result by the CCR5 cell fusion mediated, and as can be seen from the figure, maraviroc concentration is big more, and its restraining effect to the CCR5 cell fusion mediated is strong more.

Conclusion: CCR5, CXCR4 Chemokine Receptors are the important target position of development inverase; Screening system of the present invention can be used for high flux screening CCR5 or CXCR4 antagonist simply, fast, for the anti-HIV newtype drug of efficient screening provides an effective way.

SEQUENCE LISTING

< 110>Zhejiang University

< 120>a kind of screening system of HIV infected cell and application thereof

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<211>351

<212>DNA

<213>Anacystis nidulans

<400>7

gccgaatatt gtttggcggc agatacagaa gttctgaccg ttgaatatgg cccgatcgcg 60

attggcaaac tagtcgaaga aaatattcgt tgccaagttt attgctgtaa cccagatggc 120

tatatctaca gtcagccgat tggtcaatgg catcaacgag gtgaacagga agtgattgaa 180

tacgaactca gtgatggtcg catcattcga gcaactgctg accatcgctt tatgactgaa 240

gagggtgaaa tgctgtcgct ggatgaaatc tttgagcgat cgctagaact gaagcagatt 300

ccgacaccat tgttagcgat cgctcagcca tccccgttag cgacggcgta a 351

<210>8

<211>116

<212>PRT

<213>Anacystis nidulans

<400>8

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

1 5 10 15

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

20 25 30

Val Tyr Cys Cys Asn Pro Asp Gly Tyr Ile Tyr Ser Gln Pro Ile Gly

35 40 45

Gln Trp His Gln Arg Gly Glu Gln Glu Val Ile Glu Tyr Glu Leu Ser

50 55 60

Asp Gly Arg Ile Ile Arg Ala Thr Ala Asp His Arg Phe Met Thr Glu

65 70 75 80

Glu Gly Glu Met Leu Ser Leu Asp Glu Ile Phe Glu Arg Ser Leu Glu

85 90 95

Leu Lys Gln Ile Pro Thr Pro Leu Leu Ala Ile Ala Gln Pro Ser Pro

100 105 110

Leu Ala Thr Ala

115

<210>9

<211>108

<212>DNA

<213>Anacystis nidulans

<400>9

atggtcaaaa ttgttcggcg gcgttccttg ggtgtgcaac ccgtctacga ccttggcgtg 60

gcaaccgtac ataactttgt gctggccaat ggccttgtgg cctccaac 108

<210>10

<211>36

<212>PRT

<213>Anacystis nidulans

<400>10

Met Val Lys Ile Val Arg Arg Arg Ser Leu Gly Val Gln Pro Val Tyr

1 5 10 15

Asp Leu Gly Val Ala Thr Val His Asn Phe Val Leu Ala Asn Gly Leu

20 25 30

Val Ala Ser Asn

35

<210>11

<211>1065

<212>DNA

<213>Human astrovirus

<400>11

aacaagatgg attatcaagt gtcaagtcca atctatgaca tcaattatta tacatcggag 60

ccctgccaaa aaatcaatgt gaagcaaatc gcagcccgcc tcctgcctcc gctctactca 120

ctggtgttca tctttggttt tgtgggcaac atgctggtca tcctcatcct gataaactgc 180

aaaaggctga agagcatgac tgacatctac ctgctcaacc tggccatctc tgacctgttt 240

ttccttctta ctgtcccctt ctgggctcac tatgctgccg cccagtggga ctttggaaat 300

acaatgtgtc aactcttgac agggctctat tttataggct tcttctctgg aatcttcttc 360

atcatcctcc tgacaatcga taggtacctg gctgtcgtcc atgctgtgtt tgctttaaaa 420

gccaggacgg tcacctttgg ggtggtgaca agtgtgatca cttgggtggt ggctgtgttt 480

gcgtctctcc caggaatcat ctttaccaga tctcaaaaag aaggtcttca ttacacctgc 540

agctctcatt ttccatacag tcagtatcaa ttctggaaga atttccagac attaaagata 600

gtcatcttgg ggctggtcct gccgctgctt gtcatggtca tctgctactc gggaatccta 660

aaaactctgc ttcggtgtcg aaatgagaag aagaggcaca gggctgtgag gcttatcttc 720

accatcatga ttgtttattt tctcttctgg gctccctaca acattgtcct tctcctgaac 780

accttccagg aattctttgg cctgaataat tgcagtagct ctaacaggtt ggaccaagct 840

atgcaggtga cagagactct tgggatgacg cactgctgca tcaaccccat catctatgcc 900

tttgtcgggg agaagttcag aaactacctc ttagtcttct tccaaaagca cattgccaaa 960

cgcttctgca aatgctgttc tattttccag caagaggctc ccgagcgagc aagctcagtt 1020

tacacccgat ccactgggga gcaggaaata tctgtgggct tgtga 1065

<210>12

<211>354

<212>PRT

<213>Human astrovirus

<400>12

Asn Lys Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr

1 5 10 15

Tyr Thr Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gln Ile Ala Ala

20 25 30

Arg Leu Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val

35 40 45

Gly Asn Met Leu Val Ile Leu Ile Leu Ile Asn Cys Lys Arg Leu Lys

50 55 60

Ser Met Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe

65 70 75 80

Phe Leu Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gln Trp

85 90 95

Asp Phe Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Leu Tyr Phe Ile

100 105 110

Gly Phe Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg

115 120 125

Tyr Leu Ala Val Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val

130 135 140

Thr Phe Gly Val Val Thr Ser Val Ile Thr Trp Val Val Ala Val Phe

145 150 155 160

Ala Ser Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly Leu

165 170 175

His Tyr Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe Trp

180 185 190

Lys Asn Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu Val Leu Pro

195 200 205

Leu Leu Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu

210 215 220

Arg Cys Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu Ile Phe

225 230 235 240

Thr Ile Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr Asn Ile Val

245 250 255

Leu Leu Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu Asn Asn Cys Ser

260 265 270

Ser Ser Asn Arg Leu Asp Gln Ala Met Gln Val Thr Glu Thr Leu Gly

275 280 285

Met Thr His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Phe Val Gly Glu

290 295 300

Lys Phe Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys His Ile Ala Lys

305 310 315 320

Arg Phe Cys Lys Cys Cys Ser Ile Phe Gln Gln Glu Ala Pro Glu Arg

325 330 335

Ala Ser Ser Val Tyr Thr Arg Ser Thr Gly Glu Gln Glu Ile Ser Val

340 345 350

Gly Leu

<210>13

<211>1071

<212>DNA

<213>Human astrovirus

<400>13

atgtccattc ctttgcctct tttgcagata tacacttcag ataactacac cgaggaaatg 60

ggctcagggg actatgactc catgaaggaa ccctgtttcc gtgaagaaaa tgctaatttc 120

aataaaatct tcctgcccac catctactcc atcatcttct taactggcat tgtgggcaat 180

ggattggtca tcctggtcat gggttaccag aagaaactga gaagcatgac ggacaagtac 240

aggctgcacc tgtcagtggc cgacctcctc tttgtcatca cgcttccctt ctgggcagtt 300

gatgccgtgg caaactggta ctttgggaac ttcctatgca aggcagtcca tgtcatctac 360

acagtcaacc tctacagcag tgtcctcatc ctggccttca tcagtctgga ccgctacctg 420

gccatcgtcc acgccaccaa cagtcagagg ccaaggaagc tgttggctga aaaggtggtc 480

tatgttggcg tctggatccc tgccctcctg ctgactattc ccgacttcat ctttgccaac 540

gtcagtgagg cagatgacag atatatctgt gaccgcttct accccaatga cttgtgggtg 600

gttgtgttcc agtttcagca catcatggtt ggccttatcc tgcctggtat tgtcatcctg 660

tcctgctatt gcattatcat ctccaagctg tcacactcca agggccacca gaagcgcaag 720

gccctcaaga ccacagtcat cctcatcctg gctttcttcg cctgttggct gccttactac 780

attgggatca gcatcgactc cttcatcctc ctggaaatca tcaagcaagg gtgtgagttt 840

gagaacactg tgcacaagtg gatttccatc accgaggccc tagctttctt ccactgttgt 900

ctgaacccca tcctctatgc tttccttgga gccaaattta aaacctctgc ccagcacgca 960

ctcacctctg tgagcagagg gtccagcctc aagatcctct ccaaaggaaa gcgaggtgga 1020

cattcatctg tttccactga gtctgagtct tcaagttttc actccagcta a 1071

<210>14

<211>356

<212>PRT

<213>Human astrovirus

<400>14

Met Ser Ile Pro Leu Pro Leu Leu Gln Ile Tyr Thr Ser Asp Asn Tyr

1 5 10 15

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

20 25 30

Phe Arg Glu Glu Asn Ala Asn Phe Asn Lys Ile Phe Leu Pro Thr Ile

35 40 45

Tyr Ser Ile Ile Phe Leu Thr Gly Ile Val Gly Asn Gly Leu Val Ile

50 55 60

Leu Val Met Gly Tyr Gln Lys Lys Leu Arg Ser Met Thr Asp Lys Tyr

65 70 75 80

Arg Leu His Leu Ser Val Ala Asp Leu Leu Phe Val Ile Thr Leu Pro

85 90 95

Phe Trp Ala Val Asp Ala Val Ala Asn Trp Tyr Phe Gly Asn Phe Leu

100 105 110

Cys Lys Ala Val His Val Ile Tyr Thr Val Asn Leu Tyr Ser Ser Val

115 120 125

Leu Ile Leu Ala Phe Ile Ser Leu Asp Arg Tyr Leu Ala Ile Val His

130 135 140

Ala Thr Asn Ser Gln Arg Pro Arg Lys Leu Leu Ala Glu Lys Val Val

145 150 155 160

Tyr Val Gly Val Trp Ile Pro Ala Leu Leu Leu Thr Ile Pro Asp Phe

165 170 175

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

180 185 190

Phe Tyr Pro Asn Asp Leu Trp Val Val Val Phe Gln Phe Gln His Ile

195 200 205

Met Val Gly Leu Ile Leu Pro Gly Ile Val Ile Leu Ser Cys Tyr Cys

210 215 220

Ile Ile Ile Ser Lys Leu Ser His Ser Lys Gly His Gln Lys Arg Lys

225 230 235 240

Ala Leu Lys Thr Thr Val Ile Leu Ile Leu Ala Phe Phe Ala Cys Trp

245 250 255

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

260 265 270

Ile Ile Lys Gln Gly Cys Glu Phe Glu Asn Thr Val His Lys Trp Ile

275 280 285

Ser Ile Thr Glu Ala Leu Ala Phe Phe His Cys Cys Leu Asn Pro Ile

290 295 300

Leu Tyr Ala Phe Leu Gly Ala Lys Phe Lys Thr Ser Ala Gln His Ala

305 310 315 320

Leu Thr Ser Val Ser Arg Gly Ser Ser Leu Lys Ile Leu Ser Lys Gly

325 330 335

Lys Arg Gly Gly His Ser Ser Val Ser Thr Glu Ser Glu Ser Ser Ser

340 345 350

Phe His Ser Ser

355

<210>15

<211>1377

<212>DNA

<213>Human astrovirus

<400>15

atgaaccggg gagtcccttt taggcacttg cttctggtgc tgcaactggc gctcctccca 60

gcagccactc agggaaagaa agtggtgctg ggcaaaaaag gggatacagt ggaactgacc 120

tgtacagctt cccagaagaa gagcatacaa ttccactgga aaaactccaa ccagataaag 180

attctgggaa atcagggctc cttcttaact aaaggtccat ccaagctgaa tgatcgcgct 240

gactcaagaa gaagcctttg ggaccaagga aactttcccc tgatcatcaa gaatcttaag 300

atagaagact cagatactta catctgtgaa gtggaggacc agaaggagga ggtgcaattg 360

ctagtgttcg gattgactgc caactctgac acccacctgc ttcaggggca gagcctgacc 420

ctgaccttgg agagcccccc tggtagtagc ccctcagtgc aatgtaggag tccaaggggt 480

aaaaacatac agggggggaa gaccctctcc gtgtctcagc tggagctcca ggatagtggc 540

acctggacat gcactgtctt gcagaaccag aagaaggtgg agttcaaaat agacatcgtg 600

gtgctagctt tccagaaggc ctccagcata gtctataaga aagaggggga acaggtggag 660

ttctccttcc cactcgcctt tacagttgaa aagctgacgg gcagtggcga gctgtggtgg 720

caggcggaga gggcttcctc ctccaagtct tggatcacct ttgacctgaa gaacaaggaa 780

gtgtctgtaa aacgggttac ccaggaccct aagctccaga tgggcaagaa gctcccgctc 840

cacctcaccc tgccccaggc cttgcctcag tatgctggct ctggaaacct caccctggcc 900

cttgaagcga aaacaggaaa gttgcatcag gaagtgaacc tggtggtgat gagagccact 960

cagctccaga aaaatttgac ctgtgaggtg tggggaccca cctcccctaa gctgatgctg 1020

agtttgaaac tggagaacaa ggaggcaaag gtctcgaagc gggagaaggc ggtgtgggtg 1080

ctgaaccctg aggcggggat gtggcagtgt ctgctgagtg actcgggaca ggtcctgctg 1140

gaatccaaca tcaaggttct gcccacatgg tccaccccgg tgcagccaat ggccctgatt 1200

gtgctggggg gcgtcgccgg cctcctgctt ttcattgggc taggcatctt cttctgtgtc 1260

aggtgccggc accgaaggcg ccaagcagag cggatgtctc agatcaagag actcctcagt 1320

gagaagaaga cctgccagtg tcctcaccgg tttcagaaga catgtagccc catttga 1377

<210>16

<211>458

<212>PRT

<213>Human astrovirus

<400>16

Met Asn Arg Gly Val Pro Phe Arg His Leu Leu Leu Val Leu Gln Leu

1 5 10 15

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

20 25 30

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

35 40 45

Ile Gln Phe His Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn

50 55 60

Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala

65 70 75 80

Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile

85 90 95

Lys Asn Leu Lys Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu

100 105 110

Asp Gln Lys Glu Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn

115 120 125

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

130 135 140

Ser Pro Pro Gly Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly

145 150 155 160

Lys Asn Ile Gln Gly Gly Lys Thr Leu Ser Val Ser Gln Leu Glu Leu

165 170 175

Gln Asp Ser Gly Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys

180 185 190

Val Glu Phe Lys Ile Asp Ile Val Val Leu Ala Phe Gln Lys Ala Ser

195 200 205

Ser Ile Val Tyr Lys Lys Glu Gly Glu Gln Val Glu Phe Ser Phe Pro

210 215 220

Leu Ala Phe Thr Val Glu Lys Leu Thr Gly Ser Gly Glu Leu Trp Trp

225 230 235 240

Gln Ala Glu Arg Ala Ser Ser Ser Lys Ser Trp Ile Thr Phe Asp Leu

245 250 255

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

260 265 270

Gln Met Gly Lys Lys Leu Pro Leu His Leu Thr Leu Pro Gln Ala Leu

275 280 285

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

290 295 300

Thr Gly Lys Leu His Gln Glu Val Asn Leu Val Val Met Arg Ala Thr

305 310 315 320

Gln Leu Gln Lys Asn Leu Thr Cys Glu Val Trp Gly Pro Thr Ser Pro

325 330 335

Lys Leu Met Leu Ser Leu Lys Leu Glu Asn Lys Glu Ala Lys Val Ser

340 345 350

Lys Arg Glu Lys Ala Val Trp Val Leu Asn Pro Glu Ala Gly Met Trp

355 360 365

Gln Cys Leu Leu Ser Asp Ser Gly Gln Val Leu Leu Glu Ser Asn Ile

370 375 380

Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Met Ala Leu Ile

385 390 395 400

Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile

405 410 415

Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met

420 425 430

Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro

435 440 445

His Arg Phe Gln Lys Thr Cys Ser Pro Ile

450 455

<210>17

<211>2544

<212>DNA

<213>Human astrovirus

<400>17

atgagagtga aggggatcag gaagaattat cagcacttgt ggagaggggg caccttgctc 60

cttgggatgt tgatgatctg tagtgctgta gaaaaattgt gggtcacagt ctattatggg 120

gtacctgtgt ggaaagaagc aaccaccact ctattttgtg catcagatgc taaagcctat 180

gacacagagg tacataatgt ctgggccaca catgcctgtg tacccacaga ccctaaccca 240

caagaaatag tattggaaaa tgtgacagaa aattttaaca tgtggaaaaa taacatggta 300

gaacagatgc atgaggatat aatcagttta tgggatcaaa gtctaaagcc atgtgtaaag 360

ttaaccccac tctgtgttac tctacattgc actaatttga agaatgctac taataccaag 420

agtagtaatt ggaaagagat ggacagagga gaaataaaaa attgctcttt caaggtcacc 480

acaagcataa gaaataagat gcagaaagaa tatgcacttt tttataaact tgatgtagta 540

ccaatagata atgataatac aagctataaa ttgataaatt gtaacacctc agtcattaca 600

caggcctgtc caaaggtatc ctttgaacca attcccatac attattgtgc cccggctggt 660

tttgcgattc taaagtgtaa tgataagaag ttcaatggat caggaccatg tacaaatgtc 720

agcacagtac aatgtacaca tggaattagg ccagtagtgt caactcaatt gctgttaaat 780

ggcagtctag cagaagaagg ggtagtaatt agatctgaaa atttcacaga caatgctaaa 840

actataatag tacagctgaa ggaatctgta gaaattaatt gtacaagacc taacaataat 900

acaagaaaaa gtataactat aggaccgggg agagcatttt atgcaacagg agacataata 960

ggagatataa gacaagcaca ttgtaacatt agtggagaaa aatggaataa cactttaaaa 1020

cagatagtta caaaattaca agcacaattt gggaataaaa caatagtctt taagcaatcc 1080

tcaggagggg acccagaaat tgtaatgcac agttttaatt gtggagggga atttttctac 1140

tgtaattcaa cacagctttt taatagtact tggaataata ctatagggcc aaataacact 1200

aatggaacta tcacactccc atgcagaata aaacaaatta taaacaggtg gcaggaagta 1260

ggaaaagcaa tgtatgcccc tcccatcaga ggacaaatta gatgctcatc aaatattaca 1320

ggactgctat taacaagaga tggtggtaaa gagatcagta acaccaccga gatcttcaga 1380

cctggaggtg gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta 1440

aaaattgagc cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa 1500

aaaagagcag tgacgctagg agctatgttc cttgggttct tgggagcagc aggaagcact 1560

atgggcgcag cgtcactgac gctgacggta caggccagac aattattgtc tggtatagtg 1620

caacagcaga acaatttgct gagagctatt gaggcgcaac agcatctgtt gcaactcaca 1680

gtctggggca tcaagcagct ccaggcaaga gtcctggctg tggaaagata cctaaaggat 1740

caacagctcc tagggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct 1800

tggaatgcta gttggagtaa taaatctctg gatcagattt ggaataacat gacctggatg 1860

gagtgggaga gagaaattga caattacaca aacttaatat acaccttaat tgaagaatcg 1920

cagaaccaac aagaaaagaa tgaacaagaa ttattagaat tggataagtg ggcaagtttg 1980

tggaattggt ttgacatatc aaaatggctg tggtatataa aaatattcat aatgatagta 2040

ggaggtttag taggtttaag gatagttttt actgtgcttt ctatagtgaa tagagttagg 2100

cagggatact caccattatc atttcagacc cgcttcccag ccccaagggg acccgacagg 2160

cccgaaggaa tcgaagaaga aggtggagag agagacagag acagatccag tccattagtg 2220

catggattat tagcactcat ctgggacgat ctacggagcc tgtgcctctt cagctaccac 2280

cgcttgagag acttaatctt gattgcagcg aggattgtgg aacttctggg acgcaggggg 2340

tgggaagccc tcaagtattg ggggaatctc ctgcagtatt ggattcagga actaaagaat 2400

agtgctgtta gtttgtttga tgccatagct atagcagtag ctgaggggac agataggatt 2460

atagaagtag cacaaagaat tggtagagct tttctccaca tacctagaag aataagacag 2520

ggctttgaaa gggctttgct ataa 2544

<210>18

<211>847

<212>PRT

<213>Human astrovirus

<400>18

Met Arg Val Lys Gly Ile Arg Lys Asn Tyr Gln His Leu Trp Arg Gly

1 5 10 15

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

20 25 30

Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Thr

35 40 45

Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val

50 55 60

His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro

65 70 75 80

Gln Glu Ile Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys

85 90 95

Asn Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp

100 105 110

Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu

115 120 125

His Cys Thr Asn Leu Lys Asn Ala Thr Asn Thr Lys Ser Ser Asn Trp

130 135 140

Lys Glu Met Asp Arg Gly Glu Ile Lys Asn Cys Ser Phe Lys Val Thr

145 150 155 160

Thr Ser Ile Arg Asn Lys Met Gln Lys Glu Tyr Ala Leu Phe Tyr Lys

165 170 175

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

180 185 190

Asn Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val Ser Phe

195 200 205

Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala Ile Leu

210 215 220

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

225 230 235 240

Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln

245 250 255

Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Gly Val Val Ile Arg Ser

260 265 270

Glu Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln Leu Lys Glu

275 280 285

Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser

290 295 300

Ile Thr Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr Gly Asp Ile Ile

305 310 315 320

Gly Asp Ile Arg Gln Ala His Cys Asn Ile Ser Gly Glu Lys Trp Asn

325 330 335

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

340 345 350

Lys Thr Ile Val Phe Lys Gln Ser Ser Gly Gly Asp Pro Glu Ile Val

355 360 365

Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr

370 375 380

Gln Leu Phe Asn Ser Thr Trp Asn Asn Thr Ile Gly Pro Asn Asn Thr

385 390 395 400

Asn Gly Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn Arg

405 410 415

Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile Arg Gly Gln

420 425 430

Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly

435 440 445

Gly Lys Glu Ile Ser Asn Thr Thr Glu Ile Phe Arg Pro Gly Gly Gly

450 455 460

Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val

465 470 475 480

Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val

485 490 495

Val Gln Arg Glu Lys Arg Ala Val Thr Leu Gly Ala Met Phe Leu Gly

500 505 510

Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Leu Thr Leu

515 520 525

Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn

530 535 540

Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr

545 550 555 560

Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg

565 570 575

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

580 585 590

Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys

595 600 605

Ser Leu Asp Gln Ile Trp Asn Asn Met Thr Trp Met Glu Trp Glu Arg

610 615 620

Glu Ile Asp Asn Tyr Thr Asn Leu Ile Tyr Thr Leu Ile Glu Glu Ser

625 630 635 640

Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys

645 650 655

Trp Ala Ser Leu Trp Asn Trp Phe Asp Ile Ser Lys Trp Leu Trp Tyr

660 665 670

Ile Lys Ile Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile

675 680 685

Val Phe Thr Val Leu Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser

690 695 700

Pro Leu Ser Phe Gln Thr Arg Phe Pro Ala Pro Arg Gly Pro Asp Arg

705 710 715 720

Pro Glu Gly Ile Glu Glu Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser

725 730 735

Ser Pro Leu Val His Gly Leu Leu Ala Leu Ile Trp Asp Asp Leu Arg

740 745 750

Ser Leu Cys Leu Phe Ser Tyr His Arg Leu Arg Asp Leu Ile Leu Ile

755 760 765

Ala Ala Arg Ile Val Glu Leu Leu Gly Arg Arg Gly Trp Glu Ala Leu

770 775 780

Lys Tyr Trp Gly Asn Leu Leu Gln Tyr Trp Ile Gln Glu Leu Lys Asn

785 790 795 800

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

805 810 815

Thr Asp Arg Ile Ile Glu Val Ala Gln Arg Ile Gly Arg Ala Phe Leu

820 825 830

His Ile Pro Arg Arg Ile Arg Gln Gly Phe Glu Arg Ala Leu Leu

835 840 845

<210>19

<211>2571

<212>DNA

<213>Human astrovirus

<400>19

atgagagtga aggagaaata tcagcacttg tggagatggg ggtggagatg gggcaccatg 60

ctccttggga tgttgatgat ctgtagtgct acagaaaaat tgtgggtcac agtctattat 120

ggggtacctg tgtggaagga agcaaccacc actctatttt gtgcatcaga tgctaaagca 180

tatgatacag aggtacataa tgtttgggcc acacatgcct gtgtacccac agaccccaac 240

ccacaagaag tagtattggt aaatgtgaca gaaaatttta acatgtggaa aaatgacatg 300

gtagaacaga tgcatgagga tataatcagt ttatgggatc aaagcctaaa gccatgtgta 360

aaattaaccc cactctgtgt tagtttaaag tgcactgatt tgaagaatga tactaatacc 420

aatagtagta gcgggagaat gataatggag aaaggagaga taaaaaactg ctctttcaat 480

atcagcacaa gcataagagg taaggtgcag aaagaatatg cattttttta taaacttgat 540

ataataccaa tagataatga tactaccagc tataagttga caagttgtaa cacctcagtc 600

attacacagg cctgtccaaa ggtatccttt gagccaattc ccatacatta ttgtgccccg 660

gctggttttg cgattctaaa atgtaataat aagacgttca atggaacagg accatgtaca 720

aatgtcagca cagtacaatg tacacatgga attaggccag tagtatcaac tcaactgctg 780

ttaaatggca gtctagcaga agaagaggta gtaattagat ctgtcaattt cacggacaat 840

gctaaaacca taatagtaca gctgaacaca tctgtagaaa ttaattgtac aagacccaac 900

aacaatacaa gaaaaagaat ccgtatccag agaggaccag ggagagcatt tgttacaata 960

ggaaaaatag gaaatatgag acaagcacat tgtaacatta gtagagcaaa atggaataac 1020

actttaaaac agatagctag caaattaaga gaacaatttg gaaataataa aacaataatc 1080

tttaagcaat cctcaggagg ggacccagaa attgtaacgc acagttttaa ttgtggaggg 1140

gaatttttct actgtaattc aacacaactg tttaatagta cttggtttaa tagtacttgg 1200

agtactgaag ggtcaaataa cactgaagga agtgacacaa tcaccctccc atgcagaata 1260

aaacaaatta taaacatgtg gcagaaagta ggaaaagcaa tgtatgcccc tcccatcagt 1320

ggacaaatta gatgttcatc aaatattaca gggctgctat taacaagaga tggtggtaat 1380

agcaacaatg agtccgagat cttcagacct ggaggaggag atatgaggga caattggaga 1440

agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag 1500

gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt 1560

gggttcttgg gagcagcagg aagcactatg ggcgcagcct caatgacgct gacggtacag 1620

gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag 1680

gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc 1740

ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga 1800

aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa 1860

cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc 1920

ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta 1980

ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg 2040

tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct 2100

gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac 2160

ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga 2220

gacagagaca gatccattcg attagtgaac ggatccttgg cacttatctg ggacgatctg 2280

cggagcctgt gcctcttcag ctaccaccgc ttgagagact tactcttgat tgtaacgagg 2340

attgtggaac ttctgggacg cagggggtgg gaagccctca aatattggtg gaatctccta 2400

cagtattgga gtcaggaact aaagaatagt gctgttagct tgctcaatgc cacagccata 2460

gcagtagctg aggggacaga tagggttata gaagtagtac aaggagcttg tagagctatt 2520

cgccacatac ctagaagaat aagacagggc ttggaaagga ttttgctata a 2571

<210>20

<211>856

<212>PRT

<213>Human astrovirus

<400>20

Met Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg

1 5 10 15

Trp Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr Glu

20 25 30

Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala

35 40 45

Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu

50 55 60

Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn

65 70 75 80

Pro Gln Glu Val Val Leu Val Asn Val Thr Glu Asn Phe Asn Met Trp

85 90 95

Lys Asn Asp Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp

100 105 110

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

115 120 125

Leu Lys Cys Thr Asp Leu Lys Asn Asp Thr Asn Thr Asn Ser Ser Ser

130 135 140

Gly Arg Met Ile Met Glu Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn

145 150 155 160

Ile Ser Thr Ser Ile Arg Gly Lys Val Gln Lys Glu Tyr Ala Phe Phe

165 170 175

Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn Asp Thr Thr Ser Tyr Lys

180 185 190

Leu Thr Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val

195 200 205

Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala

210 215 220

Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Thr Gly Pro Cys Thr

225 230 235 240

Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser

245 250 255

Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val Ile

260 265 270

Arg Ser Val Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln Leu

275 280 285

Asn Thr Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg

290 295 300

Lys Arg Ile Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile

305 310 315 320

Gly Lys Ile Gly Asn Met Arg Gln Ala His Cys Asn Ile Ser Arg Ala

325 330 335

Lys Trp Asn Asn Thr Leu Lys Gln Ile Ala Ser Lys Leu Arg Glu Gln

340 345 350

Phe Gly Asn Asn Lys Thr Ile Ile Phe Lys Gln Ser Ser Gly Gly Asp

355 360 365

Pro Glu Ile Val Thr His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr

370 375 380

Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Phe Asn Ser Thr Trp

385 390 395 400

Ser Thr Glu Gly Ser Asn Asn Thr Glu Gly Ser Asp Thr Ile Thr Leu

405 410 415

Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys

420 425 430

Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cys Ser Ser Asn

435 440 445

Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Asn Ser Asn Asn Glu

450 455 460

Ser Glu Ile Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg

465 470 475 480

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

485 490 495

Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg Ala

500 505 510

Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser

515 520 525

Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln Leu

530 535 540

Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu

545 550 555 560

Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu

565 570 575

Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu

580 585 590

Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val

595 600 605

Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn

610 615 620

His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser

625 630 635 640

Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn

645 650 655

Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp

660 665 670

Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys Leu Phe Ile Met Ile

675 680 685

Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe Ala Val Leu Ser Ile

690 695 700

Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln Thr His

705 710 715 720

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

725 730 735

Gly Gly Glu Arg Asp Arg Asp Arg Ser Ile Arg Leu Val Asn Gly Ser

740 745 750

Leu Ala Leu Ile Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe Ser Tyr

755 760 765

His Arg Leu Arg Asp Leu Leu Leu Ile Val Thr Arg Ile Val Glu Leu

770 775 780

Leu Gly Arg Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn Leu Leu

785 790 795 800

Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu Asn

805 810 815

Ala Thr Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val

820 825 830

Val Gln Gly Ala Cys Arg Ala Ile Arg His Ile Pro Arg Arg Ile Arg

835 840 845

Gln Gly Leu Glu Arg Ile Leu Leu

850 855

Claims (9)

1. the screening system of a HIV infected cell; Comprise Chemokine Receptors screening clone and HIV envelope protein Env screening clone; Said screening system is made up by following method: (1) connects the C end of DnaE intein gene and the C end matching of reporter gene, inserts and obtains fusion expression vector 1 in the plasmid 1; The N end of DnaE intein gene and the N end matching of reporter gene are connect, insert and obtain fusion expression vector 2 in the plasmid 1; (2) the human CD 4 protein gene is inserted plasmid 2, obtain recombinant expression vector 3; Human chemokine CCR5 gene or CXCR4 gene are inserted plasmid 1, obtain recombinant expression vector 4; (3) HIV-1 virus strain Env gene HXB2-env or SF162-gp160 gene are inserted plasmid 3, obtain recombinant expression vector 5; (4) with one in carrier 1 or the carrier 2, with carrier 3 and carrier 4 cotransfections to HEK293 cell or Chinese hamster ovary celI, obtain the Chemokine Receptors screening clone of stably express; (5),, obtain the HIV envelope protein Env screening clone of stably express with carrier 5 cotransfections to Chinese hamster ovary celI or HEK293 cell with in carrier 1 or the carrier 2 another; Said reporter gene is the activated enzyme gene that is used to detect or protein gene that can be luminous; The N end matching of the C of described reporter gene end and reporter gene connects and is reporter gene, and said plasmid 1, plasmid 2, plasmid 3 are for efficiently expressing proteic carrier in mammalian cell.

2. screening system as claimed in claim 1 is characterized in that said screening system is made up by following method: (1) connects the C end of DnaE intein gene and the C end matching of reporter protein gene, inserts and obtains fusion expression vector 1 among the plasmid pcDNA3.1; The N end of DnaE intein gene and the N end matching of reporter protein gene are connect, insert and obtain fusion expression vector 2 among the plasmid pcDNA3.1; (2) people CD4 gene is inserted plasmid pFLAG-CMV ^TM-3, obtain recombinant expression vector 3; Human chemokine CCR5 or CXCR4 gene are inserted plasmid pcDNA3.1, obtain recombinant expression vector 4; (3) get HIV envelope protein gene HXB2-env and insert plasmid pSV7d or SF162-gp160 gene insertion plasmid pCAGGS, obtain recombinant expression vector 5; (4) with one in carrier 1 or the carrier 2, with carrier 3 and 4 cotransfections to HEK293 cell or Chinese hamster ovary celI, obtain the Chemokine Receptors screening clone of stably express; (5),, obtain the HIV envelope protein Env screening clone of stably express with carrier 5 cotransfections to Chinese hamster ovary celI or HEK293 cell with in carrier 1 or the carrier 2 another.

3. according to claim 1 or claim 2 screening system; The goal gene behaviour chemokine ccr 5 gene that it is characterized in that insertion in step (2) recombinant expression vector 4, the goal gene that inserts in step (3) recombinant expression vector 5 is the envelope protein SF162gp160 of HIV-1 virus strain SF162.

4. according to claim 1 or claim 2 screening system; It is characterized in that the goal gene that inserts in step (2) recombinant expression vector 4 is a human chemokine CXCR4 gene, the goal gene that inserts in step (3) recombinant expression vector 5 is the envelope protein HXB2-env of HIV-1 virus strain HXB2.

5. according to claim 1 or claim 2 screening system is characterized in that said reporter gene is one of following: 1. edge look fluorescence protein gene, 2. red fluorescent protein gene, 3. Renilla luciferase gene, 4. Firefly luciferase gene, 5. alkaline phosphatase gene, 6. Bete-lactamase gene, 7. LacZ gene.

6. according to claim 1 or claim 2 screening system is characterized in that said DnaE intein gene reads strain algae (Nostocpunctiforme) PCC73102 from basketball algae (Anacystis nidulans) R2 PCC7942 or point-like.

7. according to claim 1 or claim 2 screening system; The N end that it is characterized in that N end system clone DnaE intein gene from basketball phycomycete strain (Anacystis nidulans) R2 PCC7942 genomic dna of said DnaE intein gene; Be designated as dnae-N; The C end of the C end system of said DnaE intein gene clone DnaE intein gene from basketball phycomycete strain (Anacystis nidulans) R2 PCC7942 genomic dna is designated as dnae-C.

8. the application of screening system as claimed in claim 1 in high flux screening CCR5 or CXCR4 antagonist.

9. application as claimed in claim 8; It is characterized in that said being applied as: Chemokine Receptors is screened cell line cell and HIV envelope protein Env screening cell line cell add to add and remain in the substratum that is applicable to Chinese hamster ovary celI or HEK293 cell of screening of medicaments; Mixed culture 4～8 hours; Cultured products adds and the corresponding reaction substrate of reporter gene through washing, lysis, obtains pharmaceutical activity data to be measured.

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