CN106939319A - The host cell that a kind of HIV 1 based on mouse source cell L1210 can infect - Google Patents

The host cell that a kind of HIV 1 based on mouse source cell L1210 can infect Download PDF

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CN106939319A
CN106939319A CN201611219513.7A CN201611219513A CN106939319A CN 106939319 A CN106939319 A CN 106939319A CN 201611219513 A CN201611219513 A CN 201611219513A CN 106939319 A CN106939319 A CN 106939319A
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hiv
cell
ccr5
infect
cyclin
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曾常春
李雅婧
李丽君
何金洋
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Shenzhen Longhua District Central Hospital
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Shenzhen Longhua District Central Hospital
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Abstract

The present invention relates to applications of the mouse leukemia cell L1210 in the host cell that HIV 1 can infect is prepared, the host cell that a kind of HIV 1 can infect is further related to, it is that expression has CD4 and CCR5 on cell surface and cell inner expression has Cyclin T1 mouse leukemia cell L1210.The invention further relates to the preparation method and application of the HIV 1 host cells that can be infected.The host cell that the HIV 1 that the present invention is obtained can infect can efficiently be infected by HIV 1, and the virion replicated in infected cell has infection activity, and infected cell can be to the active viruses of HIV 1 of cell exocrine.Therefore, the host cell that HIV 1 of the invention can infect can be used for the treatment and research that HIV 1 infects, and the drug development of AntiHIV1 RT activity 1.

Description

The host cell that a kind of HIV-1 based on mouse source cell L1210 can infect
Technical field
The present invention relates to HIV therapy and research field, more specifically it relates to a kind of host cell that HIV-1 can infect and Its preparation method and application.
Background technology
Human immunodeficiency virus (Human immunodeficiency virus, HIV) is a kind of infection human immunity System cells, cause Immune Deficiency Syndrome (Acquired immunodeficiency syndrome, AIDS) Slow virus.HIV points are 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 the infection mankind and a small number of primates (such as orangutan, gibbon).Common monkey class such as perseverance The animals such as river monkey, macaque are in normal state can not infected by HIV -1.
At present, HIV-1 infection research model, mainly there is two kinds of cell model and animal model.Cell model is mainly Some CD4 positive cells, such as T4 lymphocytes, mononuclear macrophage, BMDC.These cell surfaces are with CD4 points Sub (namely HIV-1 acceptors), HIV-1 envelope protein gp120 can be combined with these CD4 molecules so that gp41 is exposed； Gp41 after exposure is combined with the cooperative expert systems CXCR4 or CCR5 on cell and is mediated HIV-1 to enter these intracellular realize and feel 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 macrophage, BMDC 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 animal model of mouse infected by HIV -1 etc..
The HIV-1 host cells research originated for human tissue, can be in the histiocytic surface loading of human origin CD4 molecules and cooperative expert systems CXCR4 or CCR5 molecule set up the cell model of HIV-1 infection, and such as TZM-bl cell lines are exactly Based on HeLa Cells, by being implanted into CD4 and CCR5 genes, make the expression CD4 and CCR5 of cell membrane surface high intensity 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 CEM, Jurkat, Hut78 and HTLV-1 infection The cell lines such as MT-2 and MT-4, can be applied to HIV-1 pathomechanism, antiviral agent by HIV-1 separation strain virus infection 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 species There is very big obstacle in specificity, progress always, such as GM95 cells, one kind is based on mouse melanoma cells, using reverse Transcribed after record viral integrase CD4, CXCR4 and CCR5 gene, make to express CD4, CXCR4 and CCR5 molecule on its cell membrane, HIV-1 infection 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, are had for the susceptible meiofauna scale-model investigations of HIV-1 Actuality meaning.
The content of the invention
L1210 cells are a kind of mouse leukemia cells, by blood disease research institute of the Chinese Academy of Sciences of China 20th century 60, One plant of Reticulocyte type leukaemia's model that the seventies is induced and set up by MLS.L1210 cells are made For a kind of neoblast of muroid haemocyte, the easy maturation in cell culture, when L1210 cells carry out murine transplanting When, either application tail vein injection, hypodermic injection or abdominal cavity injection can reach one in blood preferably L1210 cytotostatic contents, are frequently utilized for replicating the murine model of leukaemia.
However, L1210 cells but cannot function as the host cell of HIV-1 infection, because not only lacking on its cell membrane The acceptor and co-receptor molecule of HIV-1 infection, also lack the primary condition of HIV-1 virus replications.
However, it is surprising that when inventor is by CD4, CCR5 and CyclinT1 gene insertion L1210 cells in people source And make after these genes express wherein, obtained by finding the cell of genetic modification have can by the characteristic of HIV-1 virus infection, And HIV-1 viruses possess the characteristic of complete virus replication in the cell, and thus preparing one kind being capable of cross-species infection HIV-1 Rat host cell model.
Worked based on more than, the invention provides mouse leukemia cell L1210 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 that expression has CD4 and CCR5 on cell surface And cell inner expression has Cyclin T1 mouse leukemia cell L1210.
Preferably, CD4 gene expression frames, CCR5 genes are carried by being imported into the mouse leukemia cell L1210 The recombinant vector of expression cassette and Cyclin T1 gene expression frames, then screens the CD4 genes, CCR5 genes and Cyclin T1 The cell that gene is 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 including 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 L1210.
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 virus 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 into slow virus plasmid backbone and obtained.
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.
Application of the host cell that can be infected present invention also offers the HIV-1 in research HIV-1 pathogenesis.
Application of the host cell that can be infected present invention also offers the HIV-1 in anti-HIV-1 medicines 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 to the active HIV-1 viruses of cell exocrine.Cause This, the host cell that HIV-1 of the invention can infect may help to the treatment and research of HIV-1 infection, and anti-HIV-1 medicines are ground System.
Brief description of the drawings
Fig. 1 is the slow virus plasmid construct schematic diagram with hCD4/CCR5/Cyclin T1 expression cassettes；
Fig. 2 is hCD4/CCR5 mRNA real-time fluorescence quantitative PCR statistical chart；
Fig. 3 is hCD4/CCR5mRNA gel electrophoresis images；
Fig. 4 is Cyclin T1 mRNA real-time fluorescence quantitative PCR statistical charts；
Fig. 5 is hCD4/CCR5 protein immunoblot photos；
Fig. 6 is hCD4/CCR5 molecular immune image checking results；
Fig. 7 is that hCD4/CCR5/Cyclin T1 transfect L1210 cells in the PCR of HIV-1RNA in HIV-1 infected cells As a result statistical chart；
Fig. 8 is hCD4/CCR5/Cyclin T1 transfection L1210 cells HIV-1RNA in culture supernatant after HIV-1 infection PCR result statistical charts；
Fig. 9 is L1210 cells in the PCR inspections of culture supernatant HIV-1RNA infective to cem cell after HIV-1 infection Survey result statistical chart.
Embodiment
The principle and feature of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and It is non-to be used to limit the scope of the present invention.
The preparation for the host cell that 1.HIV-1 can infect
1) structure of Lentiviral
Target gene is respectively hCD4 (GenBank sequence numbers：NM_000616.4), hCCR5 (GenBank sequence numbers：NM_ 000579.3) with human Cyclin T1 (GenBank sequence numbers：NM_001240.3) it is built into double-promoter slow virus mistake Expression vector, is transferred from international standard gene pool, using bi-cistronic vectors (Bicistronic) expression way, is prepared slow Viral 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；A small amount of to extract plasmid pDown-CD4-T2A-Cyclin T1 and skeleton carrier, 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 System is supplemented to 5 μ l by the μ l of carrier 60.28ng, LR clonase 1, plus TE buffer.Add Proteinase K terminating reaction 10min.LR reaction products are converted to Stbl3, bacterium colony PCR screening positive clones, picking positive colony plasmid delivers positive colony Sequencing, obtains recombinant slow virus expression plasmid PLA.ExBi.P/puro-CMV-CD4-T2A-Cyclin T1-IRES-CCR5. CMV-CD4-T2A-Cyclin T1-IRES-CCR5 sequence such as SEQ ID NO:Shown in 1.
2) slow virus packaging produces false virus
293FT cells in good condition and in exponential phase are taken, after cell count, according to each 10cm culture Ware 5 × 106Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO2Incubator in overnight incubation；Removed before transfection in second day Old nutrient solution, add 5mL it is fresh contain 10% serum DMEM nutrient solutions.
Prepare 1 5ml centrifuge tube, first add 1.5ml serum-freesI culture mediums, add pLV/ helper-SL3、pLV/helper-SL4、pLV/helper-SL5、PLA.ExBi.P/puro-CMV-CD4-T2A-Cyclin Each 4 μ g of T1-IRES-CCR5, gently overturn and mix.It is another to take 1 5ml centrifuge tube, add 1.5ml serum-freesI is trained The Lipofectamine 2000 of nutrient solution and 40 μ l, gently overturns and mixes.It is incubated at room temperature 5min.The DNA diluted is added to Serum-free containing Lipofectamine 2000In I nutrient solutions, gently overturn and mix.Incubation at room temperature 20min, prepares the compounds of DNA-Lipofectamine 2000.
The complexes drop-wises of DNA-Lipofectamine 2000 are added in 293FT cells, put after jiggling mixing Put 37 DEG C, 5%CO2Incubated overnight in saturated humidity incubator.
What 24h replacings 10mL was fresh after transfection contains 10% serum DMEM nutrient solutions；48h after transfection, collects culture supernatant Concentrated；Change the fresh nutrient solutions of 10ml to continue to cultivate, 72h collects supernatant concentration again 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 fragment；Each centrifuge tube Fill 20ml filtrates, 50000 × g high speed centrifugation 90min precipitate virus particles, supernatant discarded；Often pipe is precipitated with 200 μ l nutrient solution weights Outstanding viral pellet, is dispensed into 2 0.5ml import AXYGEN pipes, often the μ l of pipe 100.The slow virus carrier dispensed places -80 DEG C Preserved in refrigerator.
3) slow-virus infection mouse leukemia cell L1210
L1210 cell lines come from ATCC storehouses (Number:CCL-219TM), using containing hyclone RPMI1640 culture mediums, are placed in CO2gas incubator (37 DEG C, 5%CO2) carry out cell culture.When the fusion of L1210 cells When rate is about 50% or so, slow virus 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, screened using unloaded virus, the optimal MOI of acquisition；
C, according to optimal MOI, corresponding slow virus over-express vector liquid is separately added into aim cell and control cell Each 2ml of culture medium；
CO2gas incubator (37 DEG C, 5%CO are put in after d, mixing2) 24h is incubated, complete medium culture cell is changed, A not good liquor is changed every two days up to cell is covered with, and is passed on.
4) identification of positive expression hCD4/CCR5/Cyclin T1 leukaemias
After cell line infection slow virus over-express vector culture 7-10d, the identification of related gene expression is carried out.
A, RT-PCR and detected through gel electrophoresis CD4 and CCR5 gene rna transcription, primer are as follows：
HCD4 sense primers are：5’-TGCCTCAGTATGCTGGCTCT-3’(SEQ ID NO:2)
Anti-sense primer is：5’-GAGACCTTTGCCTCCTTGTTC-3’(SEQ ID NO:3)
HCCR5 sense primer is:5’-GCTGGTCATCCTCATCCTGATAA-3’(SEQ ID NO:4)
Anti-sense 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 the 7-14 days hCD4 and hCCR5 mRNA strong positive result (P< 0.001), show after infecting slow virus carrier, L1210 cells have hCD4 and hCCR5 mRNA transcription, and transfect unloaded The result of slow virus carrier (N-T) is feminine gender.The testing result of gel electrophoresis is as shown in figure 3, specific 193bp CD4 bases The CCR5 gene segments of cause and 75bp, and unloaded control 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 using the cDNA obtained by reverse transcription as template, enters performing PCR, tested Journey is compareed with blanc cell, the unloaded body cell negative control of transfection slow virus with GADPH 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 3-14 days cyclin T1 MRNA strong positive result (P<0.001), show after infecting slow virus carrier, L1210 cells have cyclin T1 mRNA Transcription, and the result for transfecting unloaded slow virus carrier (N-T) is feminine gender.
C, immune-blotting method CD4 and CCR5 albumen expression
Extract total protein of cell；The polyacrylate hydrogel of configuration 10%, loading, race electrophoresis, transferring film, dye film, sanction film, degreasing ox Milk closing 1h, 4 DEG C of primary antibodies are incubated overnight, primary antibody 20min × 5 time, secondary antibody incubation 1h are washed with TBST, wash secondary antibody with TBST 5min × 5, luminescence imaging.
Western blot testing result is as shown in figure 5, after transfection slow virus hCD4/CCR5 over-express vectors, it is seen that 55KD CD4 albumen and 40.6KD CCR5 electroblotting These positive bands, and the unloaded control groups of N-T and blank control group are shown as negative.
D, immuno-fluorescence assay CD4 and CCR5 albumen expression
Application cell smear method is detected；PBS is washed 2 times, each 5min, with scavenger-cell nutrient solution；4% poly Formaldehyde room temperature fixes 20-30min；PBS is washed 3 times, each 5min；4%BSA room temperatures close 30min；Ratio on by specification is dilute Release after primary antibody, 4 DEG C are incubated overnight (16h)；PBS is washed 3 times, each 10min；After dilution proportion secondary antibody on by specification, lucifuge It is incubated 1h；PBS is washed 3 times, each 5min；1ug/ml DAPI lucifuges dye core 5min；PBS is washed 3 times, each 5min；Fluorescence microscope Lower observation.
As a result as shown in Figure 6, it is visible according to the fluorescent microscopic imaging result of CD4 and CCR5 molecules, as a result confirm L1210 warps After the slow virus over-express vector infection for packing hCD4/CCR5/cyclin T1, cell has the expression of CD4 and CCR5 molecules, Possess the acceptor and accessory receptor of HIV-1 viropexises infection.
2.HIV-1 is detected to the infectivity of hCD4/CCR5/Cyclin T1 murine leukemia cell L1210 cells
By hCD4/CCR5/Cyclin T1 murine leukemia cell L1210, the RPMI1640 cultures containing hyclone are incubated at In base, in CO2gas incubator (37 DEG C, 5%CO2) under cultivated, carry out HIV-1 infection cell model checking；
Pre-processed before L1210 cell infections with 2%DMSO, add 20%HIV-1 positive serums (1 × 107Cps/ml blood Final proof product) 37 DEG C of culture medium, 5% CO2gas 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 make Background control.
Cell continues to cultivate, and collects L1210 infection the 2nd, the cell of 4,6,8 days, takes the μ g of cell total rna 2 (5.5 μ l), press Its reverse transcription is cDNA by the RevertAid Reverse Transcriptase specifications of Thermo Scientific companies, Reaction condition is 42 DEG C of 1h, 75 DEG C of 5min.After 10 times of dilution, using GAPDH as reference gene, equivalent cDNA (2 μ l) is taken to be contaminated Material method RT-PCR reacts.Reaction condition is 95 DEG C of pre-degeneration 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 knot of the ratio of target gene/house-keeping gene copy number as HIV-1 RNA relative quantities Really.Same method, in the 4th, collects the supernatant of cell culture for 6,8 days, carries out HIV-1 RNA detection respectively.
As a result see Fig. 7, be as a result shown in after infected by HIV -1, the 4-6 days visible HIV-1RNA logarithm values knots of L1210 cells The obvious increase of fruit, this shows that the L1210 cells of this development can realize HIV-1 infection and there is HIV-1RNA duplication.
HIV-1RNA expressions in the supernatant of L1210 culture cells after being transfected through hCD4/CCR5/Cyclin T1 As a result Fig. 8 is seen, is as a result shown in after the 6th day there is conspicuousness HIV-1RNA in cell culture supernatant and express, and negative control The result that is then negative is organized, this shows, the L1210 cells of this development can be realized outside HIV-1 infection, can also realize that virus is multiple The characteristics of making backward cell exocrine.
The virion replicated after L1210 cell infections HIV-1 to confirm this development is active, and we are further Supernatant is infected into the cell of CEM × 174 to be tested.The cell culture supernatant and 20% without HIV-1 is applied in experiment HIV-1 positive serums (1 × 107The blood serum sample of cps/ml carrying capacity) negative and positive control is carried out respectively, in after experiment the 2nd, 3 It collects cell supernatant, and HIV-1RNA detection is carried out according to preceding method.
As a result see as shown in figure 9, the 2-3 days visible L1210 cell infections HIV-1 culture supernatant and positive controls Show HIV-1RNA conspicuousness rise.This shows that L1210 cells of the invention are secreted in culture after HIV-1 infection Virion in clear liquid is active, that is to say, that can replicate complete virus.
In brief, the HIV-1 host cells based on the white sick cell L1210 cells of mouse in the application have to HIV-1 Neurological susceptibility, and the duplication of HIV-1 intact viruses, the pathomechanism for being HIV-1, antiviral drugs, vaccine development row system can be realized For the cell model in a murine cells source new, across race.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and Within principle, any modifications, equivalent substitutions 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 mouse source cell L1210 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 (9)

1. applications of the mouse leukemia cell L1210 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 Cyclin T1 mouse leukemia cell L1210.
3. the host cell that HIV-1 according to claim 2 can infect, it is characterised in that by the white blood of the mouse The restructuring with CD4 gene expression frames, CCR5 gene expression frames and Cyclin T1 gene expression frames is imported in sick cell L1210 Carrier, then screen the cell that the CD4 genes, CCR5 genes and Cyclin T1 genes all express obtain the HIV-1 can The host cell of infection.
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 including inciting somebody to action Recombinant expression carrier with CD4 gene expression frames, CCR5 gene expression frames and Cyclin T1 gene expression frames imports described small Step in murine leukemia cell L1210.
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 virus 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, which are inserted into slow virus plasmid backbone, to be obtained.
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, Western blot.
8. application of the host cell that the HIV-1 described in Claims 2 or 3 can infect in research HIV-1 pathogenesis.
9. application of the host cell that the HIV-1 described in Claims 2 or 3 can infect in anti-HIV-1 medicines development.
CN201611219513.7A 2016-12-26 2016-12-26 The host cell that a kind of HIV 1 based on mouse source cell L1210 can infect Pending CN106939319A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113143974A (en) * 2021-03-03 2021-07-23 武汉科技大学 Method for establishing nerve injury animal model caused by HIV infection and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
SEAY, KIERAN等: "《Mice Transgenic for CD4-Specific Human CD4, CCR5 and Cyclin T1 Expression: A New Model for Investigating HIV-1 Transmission and Treatment Efficacy》", 《PLOS ONE》 *
STEFAN W等: "《NUCLEAR FACTOR kB BINDING ACTIVITY IN MOUSE L1210 CELLS FOLLOWING PHOTOFRIN II-MEDIATED PHOTOSENSITIZATION》", 《PHOTOCHERNISRRY AND PHOMBIOLOGY》 *
李雅婧等: "《表达hCD4 和hCCR5 基因的慢病毒载体的构建》", 《华夏医学》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113143974A (en) * 2021-03-03 2021-07-23 武汉科技大学 Method for establishing nerve injury animal model caused by HIV infection and application thereof
CN113143974B (en) * 2021-03-03 2023-06-06 武汉科技大学 Method for establishing HIV-infection-induced nerve injury animal model and application thereof

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Application publication date: 20170711