CN106591239A

CN106591239A - Mouse cell L615-based HIV-1 infectible host cell

Info

Publication number: CN106591239A
Application number: CN201611219588.5A
Authority: CN
Inventors: 曾常春; 李雅婧; 李丽君; 何金洋
Original assignee: Shenzhen Longhua District Central Hospital
Current assignee: Shenzhen Longhua District Central Hospital
Priority date: 2016-12-26
Filing date: 2016-12-26
Publication date: 2017-04-26

Abstract

The invention relates to an application of a mouse leukemia cell L615 in the preparation of an HIV-1 infectible host cell, and also relates to the HIV-1 infectible host cell. The mouse leukemia cell L615 is a mouse leukemia cell L615 with CD4 and CCR5 expressed on the surface of the cell and Cyclin T1 expressed in the cell. The invention also relates to a preparation method of the HIV-1 infectible host cell, and an application of the HIV-1 infectible host cell. The HIV-1 infectible host cell obtained in the invention can be highly-efficiently infected by HIV-1, virus particles replicated in the infected cell has infection activity, and the infected cell secretes active HIV-1 virus to the outside of the cell. The HIV-1 infectible host cell can be used for treating and researching HIV-1 infection and developing anti-HIV-1 drugs.

Description

The host cell that a kind of HIV-1 based on Mus source cell L615 can infect

Technical field

The present invention relates to HIV therapy and research field, more specifically it relates to a kind of host cells that can infect of HIV-1 and Its preparation method and application.

Background technology

HIV (human immunodeficiency virus) (Human immunodeficiency virus, HIV) is a kind of infection human immunity System cells, cause acquired immune deficiency syndrome (AIDS) (Acquired immunodeficiency syndrome, AIDS) Slow viruss.HIV point is 1 type and 2 types, and 1 type is the main strain of current Global prevalence, is represented with HIV-1.HIV-1 is thin to host Born of the same parents have high selectivity, are only capable of infecting the mankind and minority primate (such as orangutan, Gibbon).Common monkey class such as perseverance The animals such as river monkey, macaque are in normal state cannot infected by HIV -1.

At present, the infection research model of HIV-1, mainly there is two kinds of cell model and animal model.Cell model is mainly Some CD4 positive cells, such as T4 lymphocytes, mononuclear phagocyte, dendritic cell.These cell surfaces are with CD4 point Sub (namely HIV-1 receptors), the envelope protein gp120 of HIV-1 can be combined with these CD4 molecules so that gp41 comes out； Gp41 after exposure is combined with cooperative expert systems CXCR4 or CCR5 on cell and is mediated HIV-1 to enter these intracellular realization senses Dye；By studying reverse transcriptions and duplication situation of the HIV-1 in host cell, and then carry out the research such as anti-HIV-1 virus drugs. However, the negligible amounts that the CD4 such as T4 lymphocytes, mononuclear phagocyte, dendritic cell positive cells exist in human body, and point From difficulty.Animal model is also based on the basis of HIV-1 cell models and realizes mainly there is non-human primate model, embedding Fit Mus infected by HIV animal model etc..

For the HIV-1 host cells research in human tissue source, can be in the histiocytic surface loading of human origin Setting up the cell model of HIV-1 infection, such as TZM-bl cell lines are exactly for CD4 molecules and cooperative expert systems CXCR4 or CCR5 molecule Based on HeLa Cells, by being implanted into CD4 and CCR5 genes, the expression CD4 and CCR5 of surface of cell membrane high intensity are made Molecule, the TZM-bl cells being transformed into have height infectibility to the HIV-1 of different group varietys.Furthermore it is possible to by the mankind The work such as the screening of CD4 positive T Leukemia Cell Lines, have now been found that what CEM, Jurkat, Hut78 and HTLV-1 infected The cell lines such as MT-2 and MT-4, can separate strain virus infection by HIV-1, be applied to pathomechanism, the antiviral agents of HIV-1 In-vitro screening, vaccine research of thing etc..

However, the cell in the HIV-1 host cells in inhuman source, particularly meiofauna source, because HIV-1 is to kind of cluster , there is always very big obstacle, such as GM95 cells in specificity, one kind is based on Mus melanoma cell, using reverse in progress Transcribed after record viral integrase CD4, CXCR4 and CCR5 gene so as to CD4, CXCR4 and CCR5 molecule is expressed on cell membrane, The infection of HIV-1 can be realized, is but difficult to tie up the complete copy for continuing virus, the born of the same parents' infection that enters that can be only applied to HIV-1 is ground Study carefully.A kind of HIV-1 host cells of meiofauna derived cell are studied, for the susceptible meiofauna scale-model investigations of HIV-1 have Actuality meaning.

The content of the invention

L615 cells are a kind of mouse leukemia cells, by the Chinese Academy of Sciences of China hematopathy institute 20th century 60, One plant of Reticulocyte type leukaemia's model that the seventies is induced by mouse leukaemia virus and set up.L615 cell conducts A kind of undifferentiated cell of muroid hemocyte, it is easy to be ripe in cell culture, when L615 cells carry out murine to be transplanted, Tail vein injection, subcutaneous injection or lumbar injection are either applied, a preferable L615 can be in blood reached thin The stable content of born of the same parents, is frequently utilized for replicating leukemic murine model.

However, L615 cells but cannot function as the host cell of HIV-1 infection, because not only lacking HIV- on its cell membrane The receptor and co-receptor molecule of 1 infection, also lacks the primary condition of HIV-1 duplications.

However, it is surprising that when inventor is by CD4, CCR5 and Cyclin T1 gene insertion L615 cells in people source And after these genes is expressed wherein, the cell of genetic modification has the characteristic that can be infected by HIV-1 viruses obtained by finding, And HIV-1 viruses possess the characteristic of complete virus replication in the cell, thus preparing one kind can be across kind of a cluster infection The Rat host cell model of HIV-1.

Work based on more than, the invention provides mouse leukemia cell L615 is to prepare the host that HIV-1 can infect thin Application in born of the same parents.

Present invention also offers the host cell that a kind of HIV-1 can infect, it is characterised in that have for expression on cell surface CD4 and CCR5 and cell inner expression have the mouse leukemia cell L615 of Cyclin T1.

Preferably, by importing with CD4 gene expression frames, CCR5 gene tables in the mouse leukemia cell L615 Up to frame and the recombinant vector of Cyclin T1 gene expression frames, the CD4 genes, CCR5 genes and Cyclin T1 bases are then screened Because the cell all expressed obtains the host cell that the HIV-1 can infect.

Present invention also offers a kind of method for preparing the host cell that above-mentioned HIV-1 can infect, it is characterised in that include Recombinant expression carrier with CD4 gene expression frames, CCR5 gene expression frames and Cyclin T1 gene expression frames is imported described Step in mouse leukemia cell L615.

Preferably, the CD4 gene expression frames, CCR5 gene expression frames and Cyclin T1 gene expression frames are located at same On recombinant expression carrier, the recombinant expression carrier is slow viruss plasmid vector.

Preferably, the recombinant expression carrier is by by sequence such as SEQ ID NO:CMV-CD4-T2A- shown in 1 Cyclin T1-IRES/CCR5 are inserted in slow viruss plasmid backbone and obtain.

Preferably, the screening of the CD4 genes, CCR5 genes and Cyclin T1 gene expressions passes through RT-PCR, Diagnosis of Sghistosomiasis Mark method is carried out.

Present invention also offers application of the host cells that can infect of the HIV-1 in research HIV pathogenesis.

Present invention also offers application of the host cells that can infect of the HIV-1 in antiviral drugs development.

The host cell that the HIV-1 that the present invention is obtained can infect can efficiently be infected by HIV-1, and infected cell Middle replicated virion has infection activity, and infected cell can be viral to the activated HIV-1 of cell exocrine.Cause This, the host cell that the HIV-1 of the present invention can infect can be used for treatment and the research that HIV-1 infects, and anti-HIV-1 medicines grind System.

Description of the drawings

Fig. 1 is the slow viruss plasmid construct schematic diagram with hCD4/CCR5/Cyclin T1 expression cassettes；

Fig. 2 is the real-time fluorescence quantitative PCR cartogram of the mRNA of hCD4/CCR5；

Fig. 3 is hCD4/CCR5mRNA gel electrophoresis images；

Fig. 4 is Cyclin T1 mRNA real-time fluorescence quantitative PCR cartograms；

Fig. 5 is hCD4/CCR5 protein immunoblot photos；

Fig. 6 is hCD4/CCR5 molecular immune image checking results；

Fig. 7 is the PCR that hCD4/CCR5/Cyclin T1 transfect L615 cells HIV-1RNA in HIV-1 infected cells As a result cartogram；

Fig. 8 is that hCD4/CCR5/Cyclin T1 transfect L615 cells HIV-1RNA in culture supernatant after HIV-1 infection PCR result cartograms；

Fig. 9 is the PCR inspections of L615 cells culture supernatant HIV-1RNA infective to cem cell after HIV-1 infection Survey result cartogram.

Specific embodiment

The principle and feature of the present invention are described below in conjunction with example, example is served only for explaining the present invention, and It is non-for limiting the scope of the present invention.

The preparation of the host cell that 1.HIV-1 can infect

1) structure of Lentiviral

Genes of interest is respectively hCD4 (GenBank serial numbers：NM_000616.4), hCCR5 (GenBank serial numbers：NM_ 000579.3) with human Cyclin T1 (GenBank serial numbers：NM_001240.3 double-promoter slow viruss) are built into and cross table Up to carrier, transfer from international standard gene bank, using the expression way of bi-cistronic vectors (Bicistronic), prepare slow disease Malicious package carrier.The structural representation of this slow virus carrier is shown in Fig. 1.

Using Gateway technologies, pDown-CD4-T2A-Cyclin T1, pUP-CMV, pTail-IRES/ are built first CCR5；Plasmid pDown-CD4-T2A-Cyclin T1 and skeleton carrier are extracted in a small amount, and 25 DEG C, LR reacts 3h, and reaction system is PUP-CMV 12.89ng, pDown-CD4-T2A-Cyclin T1 10.42ng, pTail-IRES/CCR5 13.03ng, skeleton The μ l of carrier 60.28ng, LR clonase 1, plus system is supplemented to 5 μ l by TE buffer.Add E.C. 3.4.21.64 terminating reaction 10min.Conversion LR product delivers positive colony to Stbl3, bacterium colony PCR screening positive clones, picking positive colony plasmid Sequencing, obtains recombinant slow virus expression plasmid PLA.ExBi.P/puro-CMV-CD4-T2A-Cyclin T1-IRES-CCR5. The sequence of CMV-CD4-T2A-Cyclin T1-IRES-CCR5 such as SEQ ID NO:Shown in 1.

2) slow viruss packaging produces false virus

293FT cells in good condition and in exponential phase are taken, after cell counting, according to the culture of each 10cm Ware 5 × 10⁶Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO₂Incubator in overnight incubation；Remove before transfection in second day Old culture fluid, add 5mL it is fresh containing 10% serum DMEM culture fluid.

Prepare 1 5ml centrifuge tube, be initially charged 1.5ml serum-freesI culture medium, adds pLV/ helper-SL3、pLV/helper-SL4、pLV/helper-SL5、PLA.ExBi.P/puro-CMV-CD4-T2A-Cyclin The each 4 μ g of T1-IRES-CCR5, gently overturn and mix.1 5ml centrifuge tube is separately taken, 1.5ml serum-frees are addedI is trained The Lipofectamine 2000 of nutrient solution and 40 μ l, gently overturns and mixes.Incubation at room temperature 5min.The DNA for having diluted is added to Serum-free containing Lipofectamine 2000In I culture fluid, gently overturn and mix.Incubation at room temperature 20min, prepares the complex of DNA-Lipofectamine 2000.

The complexes drop-wises of DNA-Lipofectamine 2000 are added in 293FT cells, are jiggled and is put after mixing Put 37 DEG C, 5%CO₂Incubated overnight in saturated humidity incubator.

After transfection 24h change 10mL it is fresh containing 10% serum DMEM culture fluid；48h after transfection, collects culture supernatant Concentrated；Change the fresh culture fluid of 10ml to continue to cultivate, 72h collects again supernatant concentration after transfection.Method for concentration is 3000r/min low-speed centrifugal 15min, supernatant is filtered with 0.45 μ l filters, thoroughly to remove cell debriss；Each centrifuge tube Dress 20ml filtrates, 50000 × g high speed centrifugation 90min precipitate virus granules, supernatant discarded；Often pipe is precipitated with 200 μ l culture fluid weights Outstanding viral pellet, subpackage enters in 2 0.5ml import AXYGEN pipes, often the μ l of pipe 100.The good slow virus carrier of subpackage places -80 DEG C Preserve in refrigerator.

3) slow virus infection mouse leukemia cell L615

L615 cell strains are provided by the Chinese Academy of Sciences of China hematopathy institute, and RPMI1640 of the application containing hyclone is trained Foster base, is placed in CO2 gas incubator (37 DEG C, 5%CO₂) carry out cell culture.When the fusion rate of L615 cells, to be about 50% left When right, slow viruss over-express vector infection is carried out：

A, the ready culture medium of virus liquid addition for drawing exact volume (polybrene concentration is 8 μ g/ml)；

B, the unloaded virus of application are screened, and obtain optimal MOI；

C, according to optimal MOI, corresponding slow viruss over-express vector liquid is separately added in aim cell and compared with control cells The each 2ml of culture medium；

CO2 gas incubator (37 DEG C, 5%CO are put in after d, mixing₂) incubation 24h, complete medium cultured cells is changed, A not good liquor is changed every two days to cover with up to cell, is passed on.

4) identification of positive expression hCD4/CCR5/Cyclin T1 leukaemias

After cell strain infection slow viruss over-express vector culture 7-10d, the identification of related gene expression is carried out.

The rna transcription of a, RT-PCR and detected through gel electrophoresis CD4 and CCR5 genes, primer is as follows：

HCD4 forward primer is：5’-TGCCTCAGTATGCTGGCTCT-3’(SEQ ID NO:2)

Downstream primer is：5’-GAGACCTTTGCCTCCTTGTTC-3’(SEQ ID NO:3)

The forward primer of hCCR5 is:5’-GCTGGTCATCCTCATCCTGATAA-3’(SEQ ID NO:4)

Downstream primer is:5’-ATGGCCAGGTTGAGCAGGTA-3’(SEQ ID NO:5)

Cell total rna is extracted, real-time fluorescence quantitative PCR detection and detected through gel electrophoresis are carried out respectively.Real time fluorescent quantitative PCR results as shown in Fig. 2 infection slow virus carrier after 7-14 days hCD4 and hCCR5mRNA strong positive result (P< 0.001), show that Jing after infection slow virus carrier, L615 cells have the transcription of hCD4 and hCCR5mRNA, and transfect unloaded slow The result of viral vector (N-T) is feminine gender.The testing result of gel electrophoresiss is as shown in figure 3, the CD4 genes of the 193bp of specificity With the CCR5 gene segments of 75bp, and unloaded matched group (N-T) is feminine gender.

B, RT-PCR detect the rna transcription of cyclin T1 genes

Cell total rna is extracted, reverse transcription synthesis cRNA with the cDNA obtained by reverse transcription as template, enters performing PCR, tested Journey is compareed with GADPH with blanc cell, transfection slow viruss zero load somatic cell negative control as internal reference, real-time in ABI 7500 Detected on quantitative real time PCR Instrument, as a result as shown in figure 4, infection slow virus carrier after the 7-14 days cyclin T1 Strong positive result (the P of mRNA<0.001), show that L615 cells have cyclin T1 mRNA's Jing after infection slow virus carrier Transcription, and the result for transfecting unloaded slow virus carrier (N-T) is feminine gender.

The expression of c, immune-blotting method CD4 and CCR5 albumen

Extract total protein of cell；The polyacrylate hydrogel of configuration 10%, loading, race electrophoresis, transferring film, dye film, sanction film, defat cattle Milk closing 1h, 4 DEG C of one anti-overnight incubation, wash anti-20min × 5 time, two anti-incubation 1h with TBST, that two are washed with TBST is anti- 5min × 5, luminescence imaging.

Immunoblotting testing result is as shown in figure 5, after transfection slow viruss hCD4/CCR5 over-express vectors, it is seen that 55KD CD4 albumen and 40.6KD CCR5 electroblotting These positive bands, and N-T zero load matched groups and blank control group are shown as negative.

The expression of d, immuno-fluorescence assay CD4 and CCR5 albumen

Application cell smear method is detected；PBS is washed 2 times, each 5min, with scavenger cell culture fluid；4% poly first Aldehyde room temperature fixes 20-30min；PBS is washed 3 times, each 5min；4%BSA room temperatures close 30min；Dilution proportion on by specification After one is anti-, 4 DEG C of overnight incubations (16h)；PBS is washed 3 times, each 10min；After dilution proportion two on by specification resists, lucifuge is incubated Educate 1h；PBS is washed 3 times, each 5min；1ug/ml DAPI lucifuges contaminate core 5min；PBS is washed 3 times, each 5min；Under fluorescence microscope Observation.

As a result as shown in fig. 6, the fluorescent microscopic imaging result of CD4 and CCR5 molecules confirms the packaged hCD4/CCR5/ of L615 After the slow viruss over-express vector infection of cyclin T1, cell has the expression of CD4 and CCR5 molecules, possesses HIV-1 viral Enter the receptor and accessory receptor of born of the same parents' infection.

Infectivity detections of the 2.HIV-1 to hCD4/CCR5/Cyclin T1 murine leukemia cell L615 cells

By hCD4/CCR5/Cyclin T1 murine leukemia cell L615, the cultures of the RPMI1640 containing hyclone are incubated at In base, in CO2 gas incubator (37 DEG C, 5%CO₂) under cultivated, carry out HIV-1 infection cell model checking；

2%DMSO pretreatment is used before L615 cell infections, 20%HIV-1 positive serums (1 × 10 are added⁷The blood of cps/ml Final proof product) 37 DEG C of culture medium, 5% CO2 gas incubator be incubated 12 hours with infection cell；The culture medium or PBS of serum-free Cleaning 2 times, plus normal complete medium, the part culture supernatant left and taken now makees Background control.

Cell continues to cultivate, and collects the cell that L615 infects the 4th, 6 days, takes the μ g of cell total rna 2 (5.5 μ l), presses Its reverse transcription is cDNA by the RevertAid Reverse Transcriptase description of Thermo Scientific companies, Reaction condition is 42 DEG C of 1h, 75 DEG C of 5min.After 10 times of dilution, with GAPDH as reference gene, take equivalent cDNA (2 μ l) and contaminated Material method RT-PCR is reacted.Reaction condition is 95 DEG C of denaturations 1min；95 DEG C of 15s, 60 DEG C of 1min, 40 circulations；Solubility curve 95 DEG C 15s, 60 DEG C of 30s, 95 DEG C of 15s.With the ratio of genes of interest/house-keeping gene copy number as HIV RNA relative quantities result. Same method, in the 2nd, 4,6 days intervals the supernatant of cell culture is collected, and carries out the detection of HIV RNA.

As a result see Fig. 7, be as a result shown in after infected by HIV -1, the 4-6 days visible HIV-1RNA relative values knots of L615 cells The obvious increase of fruit, this shows that the L615 cells of this development can realize the infection of HIV-1 and there is the duplication of HIV-1RNA.

HIV-1RNA expressions knot in the supernatant of the L615 cultured cells Jing after hCD4/CCR5/Cyclin T1 transfections Fruit sees Fig. 8, after being as a result shown in the 6th day, there is significance HIV-1RNA in cell culture supernatant and expresses, and negative control group Then be negative result, and this shows, the L615 cells of this development can be realized outside the infection of HIV-1, can also realize virus replication The characteristics of backward cell exocrine.

Virion to be replicated after the L615 cell infection HIV-1 for confirming this development is active, and we are further Supernatant is infected into CEM × 174 cell to be tested.Using the cell culture supernatant and 20% without HIV-1 in experiment HIV-1 positive serums (1 × 10⁷The blood serum sample of cps/ml carrying capacity) feminine gender and positive control are carried out respectively, the 2nd, 3 after experiment It collects cell supernatant, and according to front method the detection of HIV-1RNA is carried out.

As a result culture supernatant and positive controls as shown in figure 9, the 2-3 days visible L615 cell infections HIV-1 are seen The significance for showing HIV-1RNA is raised.This shows that the L615 cells of the present invention are secreted in culture supernatant after the infection of HIV-1 Virion in liquid is active, that is to say, that can replicate complete virus.

In brief, in the application HIV-1 is had easily based on the HIV-1 host cells of mice white disease cell L615 cells Perception, and the duplication of HIV-1 intact viruses can be realized, it is pathomechanism, antiviral drugs, the preparation of vaccine development row of HIV-1 The cell model in one murine cells source new, across race.

The foregoing is only presently preferred embodiments of the present invention, not to limit the present invention, all spirit in the present invention and Within principle, any modification, equivalent substitution and improvements made etc. should be included within the scope of the present invention.

Sequence table

<110>Shenzhen Longhua new district central hospital

<120>The host cell that a kind of HIV-1 based on Mus source cell L615 can infect

<130> 1

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 5899

<212> DNA

<213>Artificial sequence

<400> 1

tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540

acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc aagtttgtac 600

aaaaaagcag gctgccacca tgaaccgggg agtccctttt aggcacttgc ttctggtgct 660

gcaactggcg ctcctcccag cagccactca gggaaagaaa gtggtgctgg gcaaaaaagg 720

ggatacagtg gaactgacct gtacagcttc ccagaagaag agcatacaat tccactggaa 780

aaactccaac cagataaaga ttctgggaaa tcagggctcc ttcttaacta aaggtccatc 840

caagctgaat gatcgcgctg actcaagaag aagcctttgg gaccaaggaa actttcccct 900

gatcatcaag aatcttaaga tagaagactc agatacttac atctgtgaag tggaggacca 960

gaaggaggag gtgcaattgc tagtgttcgg attgactgcc aactctgaca cccacctgct 1020

tcaggggcag agcctgaccc tgaccttgga gagcccccct ggtagtagcc cctcagtgca 1080

atgtaggagt ccaaggggta aaaacataca gggggggaag accctctccg tgtctcagct 1140

ggagctccag gatagtggca cctggacatg cactgtcttg cagaaccaga agaaggtgga 1200

gttcaaaata gacatcgtgg tgctagcttt ccagaaggcc tccagcatag tctataagaa 1260

agagggggaa caggtggagt tctccttccc actcgccttt acagttgaaa agctgacggg 1320

cagtggcgag ctgtggtggc aggcggagag ggcttcctcc tccaagtctt ggatcacctt 1380

tgacctgaag aacaaggaag tgtctgtaaa acgggttacc caggacccta agctccagat 1440

gggcaagaag ctcccgctcc acctcaccct gccccaggcc ttgcctcagt atgctggctc 1500

tggaaacctc accctggccc ttgaagcgaa aacaggaaag ttgcatcagg aagtgaacct 1560

ggtggtgatg agagccactc agctccagaa aaatttgacc tgtgaggtgt ggggacccac 1620

ctcccctaag ctgatgctga gcttgaaact ggagaacaag gaggcaaagg tctcgaagcg 1680

ggagaaggcg gtgtgggtgc tgaaccctga ggcggggatg tggcagtgtc tgctgagtga 1740

ctcgggacag gtcctgctgg aatccaacat caaggttctg cccacatggt ccaccccggt 1800

gcagccaatg gccctgattg tgctgggggg cgtcgccggc ctcctgcttt tcattgggct 1860

aggcatcttc ttctgtgtca ggtgccggca ccgaaggcgc caagcagagc ggatgtctca 1920

gatcaagaga ctcctcagtg agaagaagac ctgccagtgc cctcaccggt ttcagaagac 1980

atgtagcccc attgagggca ggggaagtct tctaacatgc ggggacgtgg aggaaaatcc 2040

cggccccatg gagggagaga ggaagaacaa caacaaacgg tggtatttca ctcgagaaca 2100

gctggaaaat agcccatccc gtcgttttgg cgtggaccca gataaagaac tttcttatcg 2160

ccagcaggcg gccaatctgc ttcaggacat ggggcagcgt cttaacgtct cacaattgac 2220

tatcaacact gctatagtat acatgcatcg attctacatg attcagtcct tcacacagtt 2280

ccctggaaat tctgtggctc cagcagcctt gtttctagca gctaaagtgg aggagcagcc 2340

caaaaaattg gaacatgtca tcaaggtagc acatacttgt ctccatcctc aggaatccct 2400

tcctgatact agaagtgagg cttatttgca acaagttcaa gatctggtca ttttagaaag 2460

cataattttg cagactttag gctttgaact aacaattgat cacccacata ctcatgtagt 2520

aaagtgcact caacttgttc gagcaagcaa ggacttagca cagacttctt acttcatggc 2580

aaccaacagc ctgcatttga ccacatttag cctgcagtac acacctcctg tggtggcctg 2640

tgtctgcatt cacctggctt gcaagtggtc caattgggag atcccagtct caactgacgg 2700

gaagcactgg tgggagtatg ttgacgccac tgtgaccttg gaacttttag atgaactgac 2760

acatgagttt ctacagattt tggagaaaac tcccaacagg ctcaaacgca tttggaattg 2820

gagggcatgc gaggctgcca agaaaacaaa agcagatgac cgaggaacag atgaaaagac 2880

ttcagagcag acaatcctca atatgatttc ccagagctct tcagacacaa ccattgcagg 2940

tttaatgagc atgtcaactt ctaccacaag tgcagtgcct tccctgccag tctccgaaga 3000

gtcatccagc aacttaacca gtgtggagat gttgccgggc aagcgttggc tgtcctccca 3060

accttctttc aaactagaac ctactcaggg tcatcggact agtgagaatt tagcacttac 3120

aggagttgat cattccttac cacaggatgg ttcaaatgca tttatttccc agaagcagaa 3180

tagtaagagt gtgccatcag ctaaagtgtc actgaaagaa taccgcgcga agcatgcaga 3240

agaattggct gcccagaaga ggcaactgga gaacatggaa gccaatgtga agtcacaata 3300

tgcatatgct gcccagaatc tcctttctca tcatgatagc cattcttcag tcattctaaa 3360

aatgcccata gagggttcag aaaaccccga gcggcctttt ctggaaaagg ctgacaaaac 3420

agctctcaaa atgagaatcc cagtggcagg tggagataaa gctgcgtctt caaaaccaga 3480

ggagataaaa atgcgcataa aagtccatgc tgcagctgat aagcacaatt ctgtagagga 3540

cagtgttaca aagagccgag agcacaaaga aaagcacaag actcacccat ctaatcatca 3600

tcatcatcat aatcaccact cacacaagca ctctcattcc caacttccag ttggtactgg 3660

gaacaaacgt cctggtgatc caaaacatag tagccagaca agcaacttag cacataaaac 3720

ctatagcttg tctagttctt tttcctcttc cagttctact cgtaaaaggg gaccctctga 3780

agagactgga ggggctgtgt ttgatcatcc agccaagatt gccaagagta ctaaatcctc 3840

ttccctaaat ttctccttcc cttcacttcc tacaatgggt cagatgcctg ggcatagctc 3900

agacacaagt ggcctttcct tttcacagcc cagctgtaaa actcgtgtcc ctcattcgaa 3960

actggataaa gggcccactg gggccaatgg tcacaacacg acccagacaa tagactatca 4020

agacactgtg aatatgcttc actccctgct cagtgcccag ggtgttcagc ccactcagcc 4080

cactgcattt gaatttgttc gtccttatag tgactatctg aatcctcggt ctggtggaat 4140

ctcctcgaga tctggcaata cagacaaacc ccggccacca cctctgccat cagaacctcc 4200

tccaccactt ccaccccttc ctaagtaaac ccagctttct tgtacaaagt gggcccctct 4260

ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt 4320

gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct 4380

ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa 4440

ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg 4500

tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc 4560

caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg 4620

agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg 4680

aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtacacatgc 4740

tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg 4800

gttttccttt gaaaaacacg atgataatat ggccacaacc atggattatc aagtgtcaag 4860

tccaatctat gacatcaatt attatacatc ggagccctgc caaaaaatca atgtgaagca 4920

aatcgcagcc cgcctcctgc ctccgctcta ctcactggtg ttcatctttg gttttgtggg 4980

caacatgctg gtcatcctca tcctgataaa ctgcaaaagg ctgaagagca tgactgacat 5040

ctacctgctc aacctggcca tctctgacct gtttttcctt cttactgtcc ccttctgggc 5100

tcactatgct gccgcccagt gggactttgg aaatacaatg tgtcaactct tgacagggct 5160

ctattttata ggcttcttct ctggaatctt cttcatcatc ctcctgacaa tcgataggta 5220

cctggctgtc gtccatgctg tgtttgcttt aaaagccagg acggtcacct ttggggtggt 5280

gacaagtgtg atcacttggg tggtggctgt gtttgcgtct ctcccaggaa tcatctttac 5340

cagatctcaa aaagaaggtc ttcattacac ctgcagctct cattttccat acagtcagta 5400

tcaattctgg aagaatttcc agacattaaa gatagtcatc ttggggctgg tcctgccgct 5460

gcttgtcatg gtcatctgct actcgggaat cctaaaaact ctgcttcggt gtcgaaatga 5520

gaagaagagg cacagggctg tgaggcttat cttcaccatc atgattgttt attttctctt 5580

ctgggctccc tacaacattg tccttctcct gaacaccttc caggaattct ttggcctgaa 5640

taattgcagt agctctaaca ggttggacca agctatgcag gtgacagaga ctcttgggat 5700

gacgcactgc tgcatcaacc ccatcatcta tgcctttgtc ggggagaagt tcagaaacta 5760

cctcttagtc ttcttccaaa agcacattgc caaacgcttc tgcaaatgct gttctatttt 5820

ccagcaagag gctcccgagc gagcaagctc agtttacacc cgatccactg gggagcagga 5880

aatatctgtg ggcttgtga 5899

<210> 2

<211> 20

<212> DNA

<213>Artificial sequence

<400> 2

tgcctcagta tgctggctct 20

<210> 3

<211> 21

<212> DNA

<213>Artificial sequence

<400> 3

gagacctttg cctccttgtt c 21

<210> 4

<211> 23

<212> DNA

<213>Artificial sequence

<400> 4

gctggtcatc ctcatcctga taa 23

<210> 5

<211> 20

<212> DNA

<213>Artificial sequence

<400> 5

atggccaggt tgagcaggta 20

Claims

1. applications of the mouse leukemia cell L615 in the host cell that HIV-1 can infect is prepared.

2. the host cell that a kind of HIV-1 can infect, it is characterised in that have CD4 and CCR5 and thin for expression on cell surface Intracellular expression has the mouse leukemia cell L615 of Cyclin T1.

3. the host cell that HIV-1 according to claim 2 can infect, it is characterised in that by the white blood of the mice Import the restructuring with CD4 gene expression frames, CCR5 gene expression frames and Cyclin T1 gene expression frames in sick cell L615 to carry Body, then screening the cell that the CD4 genes, CCR5 genes and Cyclin T1 genes all express and obtaining the HIV-1 to feel The host cell of dye.

4. a kind of method for preparing the host cell that HIV-1 as claimed in claim 3 can infect, it is characterised in that include by Recombinant expression carrier with CD4 gene expression frames, CCR5 gene expression frames and Cyclin T1 gene expression frames imports described little Step in murine leukemia cell L615.

5. method according to claim 4, it is characterised in that the CD4 gene expression frames, CCR5 gene expression frames and Cyclin T1 gene expression frames are located on same recombinant expression carrier, and the recombinant expression carrier is slow viruss plasmid vector.

6. method according to claim 5, it is characterised in that the recombinant expression carrier is by by sequence such as SEQ ID NO:CMV-CD4-T2A-Cyclin T1-IRES/CCR5 shown in 1 are inserted in slow viruss plasmid backbone and obtain.

7. according to the method any one of claim 4-6, it is characterised in that the CD4 genes, CCR5 genes and Cyclin The screening of T1 gene expressions is carried out by RT-PCR, immunoblotting.

8. application of the host cell that the HIV-1 described in Claims 2 or 3 can infect in research HIV pathogenesis.

9. application of the host cell that the HIV-1 described in Claims 2 or 3 can infect in antiviral drugs development.