CN108103095A - The gene targeting carrier and construction method of a kind of AntiHIV1 RT activity/AIDS and application - Google Patents
The gene targeting carrier and construction method of a kind of AntiHIV1 RT activity/AIDS and application Download PDFInfo
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Abstract
The invention belongs to bioengineering fields, and in particular to build and obtain the targeting vector that the regulation type ZFN Expression elements fixed point insertion HIV 1 with targeting excision 1 provirus genes of HIV is infected to auxiliary receptor CCR 5 site；Human hematopoietic stem cell or immunocyte through the carrier modification, the infection of CCR5 preferendum strains can be blocked by being not only due to the knockout of CCR5 molecules, and, also the provirus gene of CXCR4 preferendums strain or the virus leak into, latent infection in host target cells chromosome can be cut off, there is more thorough and extensive AntiHIV1 RT activity infection activity.
Description
Technical field
The invention belongs to bioengineering field, it is related to gene targeting carrier and the structure side of a kind of new AntiHIV1 RT activity/AIDS
Method, and in particular in the targeting vector and structure of the site-directed insertion AIDS virus resisting elements of CCR5, more particularly, to target
Auxiliary receptor CCR 5 is infected to HIV-1 viruses are knocked out and in the site-directed targeting vector for being inserted into antiviral element and its preparation
Method.
Background technology
Prior art discloses AIDS (Acquired immunodeficiency syndrome, AIDS) mainly by
A kind of infection caused by human immunodeficiency virus I types (Human immunodeficiency virus 1, HIV-1) infection
Property disease.Practice display, although, highly effective antiretroviral therapy (Highly active antiretroviral
Therapy, HAART) can be with suppressing virus replication, extension patient's service life, but certain deficiency is still had in use：Such as
Expensive consumption cost, prolonged administration of drugs with side effect, virus drug resistance and current antiviral drug cannot eradicate
A series of problems, such as viral.So far, AIDS vaccine research has two more than ten years in the world, although consumption huge fund has carried out this
More than 100 clinical test of class vaccine, but at present effective vaccine there is no to come out, two clinical tests for completing for III phase are with mistake
It loses and comes to an end, so the finding the product for the treatment of of AIDS of the task is heavier.
2007, after " Berlin patient " receives Bone Marrow Stem Cells Transplantation in Treating, AIDS virus was from the miracle to disappear in vivo
Give the new hope of people.Researcher discloses, and the distribution type of the reason for patient successfully cures not only is only that bone marrow donor person is very
It coincide, and also has a kind of mutant gene CCR5 that can naturally resist AIDS virus in the marrow donated.According to statistics, this change
Allogene only exists in a small number of West Europe crowds, and therefore, searching distribution type is coincide and the donor of CCR5 gene natural variations belongs to tired in fact
It is difficult.But, the example prompting that " Berlin " patient successfully cures using virus infection auxiliary receptor CCR 5 molecule as target spot, can block
The infection of virus, the orientation treatment of AIDS has been introduced into field of gene by this.
In recent years, gene editing technology presents certain advantage in terms of the gene therapy of human diseases, undoubtedly, this
Technology also shows advantage in the gene therapy of AIDS.2008, Perez etc. carried zinc finger by adenovirus (Ad5/35 types)
Nuclease (Zinc finger nucleases, ZFNs) primary CD4 of targeting knock out+CCR5 genes in T cell simultaneously repair gene
CD4 after decorations+T cell is fed back in Mice Body, shows apparent antivirus action (Perez EE, et
al.Establishment ofHIV-1resistance in CD4+T cells by genome editing using
zinc-finger nucleases.Nat Biotechnol.2008,26(7):808-816).The AntiHIV1 RT activity of this technology mediation/
The treatment of AIDS has been enter into clinical trial (NCT00842634), and 2010, Holt etc. was imported ZFNs by the rotaring transfecting mode that shocks by electricity
Antiviral activity detection is carried out in human hematopoietic stem cell (Hematopoietic stem cells, HSCs) and in vivo, tied
Fruit shows that immunocyte can be rebuild in the lymphoid organ of mouse, and compared with unmodified group, the HSCs modified through ZFNs is shown
Good antiviral effect (Holt N, et al.Human hematopoietic stem/progenitor cells
modified by zinc-finger nucleases targeted to CCR5control HIV-1in
vivo.Nat.Biotechnol,2010,28(8):839-847)；2013, Pablo etc. utilized mature targeting CCR5's
ZFNs (SB-728-T) is to the CD4 of AIDS patient+T cell is modified for cell autograft, the results show that 6
Name is discontinued in the AIDS patient for the treatment of, and the in vivo virus load of 4 people is reduced, and viral load is nearly no detectable in 1 human body
Measure (Tebas P, et al.Gene editing of CCR5 in autologous CD4 T cells of persons
infected with HIV.N Engl J Med,2014,370(10):901-910), which shows, to T cell
Transformation can make AIDS patient from the long-term treatment of drug；Meanwhile the paces of energy propulsion functions treatment of AIDS.But
Above-mentioned means of intervention still has certain shortcoming：(1) the T cell needs through ZFNs modifications feed back, are time-consuming longer in vivo；
(2) ZFNs modification T cell only have resistant function to R5 tropics, to X4 tropics (using CXCR4 molecules for aid in by
Body) but passivity；(3) T cell through ZFNs modifications can be with the infection of blocking virus, but to seize the opportunity entrance or latent infection
Provirus in host chromosome is helpless, and these viral presence will certainly cause the sense again of HIV-1 viruses
Dye.More than reason is based on, this field researcher thinks, the sense of X4 tropics can not be blocked by only knocking out CCR5 molecules
A kind of dye, it is also necessary to new wider antiviral means.
It designs before the seminar of the applicant and obtains a pair of of special target majority HIV-1 subtype genes conserved region
The zinc fingers nuclease (Zinc-finger-nucleases, ZFN) of LTR, in virus lays dormant infection and infection cell model
It confirms that ZFN special target HIV-1LTR and can efficiently cut off the overall length HIV-1 provirus genes of integration, achieves effective anti-
Viral infectious effect (Qu X, et al.Zinc-finger-nucleases mediate specific and efficient
excision of HIV-1proviral DNA from infected and latently infected human T
cells.Nucleic Acids Res.2013,41(16):7771-7782).Based on this, present inventor intends providing one
The gene targeting carrier and construction method of the new AntiHIV1 RT activity/AIDS of kind, especially in the site-directed insertion AIDS virus resistings of CCR5
The targeting vector of element makes realization ZFN targetings cut off virus genomic purpose and solve to dive caused by long-term expression ZFN
In genotoxicity issues.
The content of the invention
It is an object of the invention to the present situations based on the prior art, intend providing a kind of gene targeting of new AntiHIV1 RT activity/AIDS
Carrier and construction method, and in particular to CCR5 it is site-directed insertion AIDS virus resisting element targeting vector and structure, especially
It is related to for targeting knock out HIV-1 viruses infection auxiliary receptor CCR 5 and in the target practice of the site-directed antiviral element of insertion
Carrier and its construction method.
The purpose of the present invention is achieved through the following technical solutions,
The ZFN fixed points for targeting virus LTR are inserted into while CCR5 molecules are knocked out and knock out site, reaches and refuses virus
Outdoors, and remove and seize the opportunity into the virus CXCR4 Strain that forms of development and latent infection in host chromosome genome
HIV-1 provirus genes purpose；In addition, structure regulation type ZFN, the protein induced ZFN of Tat expressed after being infected by virus
Expression, solve the problems, such as that latent gene misses the target caused by ZFN continuous expressions, realize that ZFN targetings cut off virus genomic mesh
, and latent gene toxicity problem caused by solving long-term expression ZFN.
It,, will while CCR5 molecules are knocked out by the nuclease-mediated homologous recombination approach of gene editing in the present invention
Target virus LTR the insertion of regulation type ZFN orientations its destroy site, acquisition one kind can targeting knock out CCR5 molecules but also will
The gene targeting carrier of new AntiHIV1 RT activity/AIDS of the HIV-1 provirus gene excisions of integration.
More specifically, the present invention provides target virus for targeting knock out CCR5 molecules and destroying site-directed integrate
The gene targeting carrier of the regulation type ZFN of gene LTR, i.e. pCCR5 donor-LTR-ZFN plasmids；The plasmid is with targeting CCR5's
Gene editing support C RISPR/Cas9 is transferred into the cell jointly, is occurred in the DNA incisions that restriction endonuclease CRISPR/Cas9 is generated
Orient homologous recombination；CCR5 knockouts site can be integrated in by targeting the regulation type ZFN Expression elements of LTR as a result,；Once HIV-1 is sick
Malicious target cell infection, will express Tat albumen and trans-activation targeting LTR ZFN expression, so as to by 5 ' LTR and 3 ' LTR it
Between proviral DNA cut off.
In the present invention, involved recognition site CCR5 target practices site is from research report (Lin Ye, et
al.Seamless modification of wild-type induced pluripotent stem cells to the
natural CCR5Δ32mutation confers resistance to HIV infection.Proc Natl Acad
Sci USA.2014,111(26):9591-9596)；Recognition site sequence is CATACAGTCAGTATCAATTCTGG.
In the present invention, plasmid construction includes：Targeting is being inserted among the upstream and downstream homology arm of CCR5 genes for knocking out
The ZFN expressed sequences of LTR and the riddled basins MGMTP140K Expression elements for being enriched with genetically modified cell, the target practice
Carrier can be built by art methods, be named as pCCR5 donor-LTR-ZFN；In the present invention, at the same construct for pair
According to gene targeting carrier, i.e., be only inserted into be enriched with the selection markers base of genetically modified cell among the homology arm of upstream and downstream
Because of Expression element, pCCR5 donor-MGMTP140K are named as.
The present invention has carried out the genetic recombination detection of gene targeting carrier：It is thin by test of human embryonic kidney cells HEK293 cells
Born of the same parents turn the gene editing support C RISPR/Cas9 for targeting CCR5 and gene targeting carrier pCCR5 donor-LTR-ZFN jointly
Dye five days after transfection, is screened, for being enriched with the thin of genetic modification into HEK293 cells through BCNU/BG dual drugs
Born of the same parents；The mdr cell finally obtained is expanded into culture and the genetic recombination situation of targeting vector is detected by PCR modes；Wherein,
The genome in genetically modified cell is extracted, upstream and downstream site-directed integration product is expanded by specific primer；PCR the results show that
Corotation is infected in the HEK293 cells of gene editing carrier and gene targeting carrier, is detected upper and lower in gene targeting carrier
Expected genetic recombination can occur for trip homology arm, and it is respectively 737bp and 1053bp to integrate band, with expected stripe size one
It causes；It is the ideal sequence after genetic recombination for the band that further has a definite purpose, continues pcr amplification product being connected into the general load of sequencing
Sequence verification is carried out in body pMD18-T, the results show that institute's amplified band is genetic recombination sequence really.
The present invention has carried out the bioactivity detection of antiviral element：CCR5 knockout integrations there is into antiviral element
HEK293 cells (ZFN) are only integrated with the HEK293 cell inoculations of riddled basins (MGMTP140K) in 24 orifice plates, so
The HIV-1 virus particles pHIV-NL4-3-luc of luciferase reporter gene luciferase and reference gene table will be carried afterwards
It is transfected up to plasmid pRL-SV40pA into above two cell line；Three days after transfection, supernatant was used for by harvest cell, cracking
The detection of uciferase activity, testing result show that relative incorporation has the cell line of screening-gene, are being integrated with the cell of ZFN
In system, the relative activity of luciferase significantly decreases, and shows that ZFN has certain bioactivity (as shown in Figure 4)；For into
One step demonstrate,proves the bioactivity of ZFN, continues with the false type HIV-1 virus infection above two cells for carrying GFP reporter genes
System, it is metainfective 72 it is small when, changed by GFP positive cell numbers purpose in flow cytomery different disposal group, as a result
Display after virus infects, compared with cellular control unit system, is integrated with GFP positive cells in the cell line of the antiviral elements of ZFN
Number has 11% decline, it was demonstrated that ZFN has certain bioactivity.
The present invention has carried out the detection of expression after CCR5 molecules knock out：It is thin to express the TZM-bl of CCR5 and CXCR4 molecules
Born of the same parents are target cell, by transiently transfecting mode by gene editing support C RISPR/Cas9 and gene targeting carrier pCCR5
Donor-LTR-ZFN or pCCR5 donor-MGMTP140K are directed into jointly in TZM-bl cells, the 5th after transfection day, are led to
It crosses BCNU/BG drugs and carries out screening acquisition mdr cell, the mdr cell of acquisition is expanded culture and passes through flow cytometer and is examined
Survey the variation of CCR5 expression quantity；It is analyzed through flow cytomery, compared with unmodified TZM-bl cells (Mock), transfection has
The TZM-bl cells (being named as MGMTP140K) or CRISPR/Cas9 of CRISPR/Cas9 and pCCR5 donor-MGMTP140K
It is decreased obviously with the CCR5 expression quantity in the TZM-bl cells (being named as ZFN) of pCCR5 donor-LTR-ZFN.
The present invention has carried out the antiviral activity detection of genetically modified cell TZM-bl：Based on because being integrated in TZM-bl cells
Have viral promotors LTR regulate and control luciferase reporter gene, once virus infection, will induced fluorescence element enzyme expression；This
In invention, the TZM-bl cells (MGMTP140K and ZFN) of CCR5 gene knockouts and unmodified TZM-bl cells (Mock) are connect
Kind is in 24 orifice plates, next day, infects above-mentioned cell with R5 or X4 tropic strains, when 37 DEG C of placements 2 are small, afterwards infects virus
Liquid removes, and is replaced with complete medium and continues to cultivate, after three days, collects cell and crack, and gained supernatant is used for luciferase
Determination of activity；It is analyzed through luciferase reporter gene detecting system, after the infection of R5 tropic strains, CCR5 genetic modifications
Luciferase expression amount intracellular TZM-bl is significantly lower than unmodified TZM-bl cells, integrated therein with the antiviral members of ZFN
The luciferase expression amount that the TZM-bl of part is intracellular is minimum；After the infection of X4 tropics, the antiviral elements of ZFN are only integrated with
The intracellular luciferase expressions of TZM-bl decline；The above results confirm that knocking out integrations in CCR5 has antiviral element
The modified cells of ZFN can carry out dual resistance to R5 preferendums and X4 tropics；CCR5 molecules are only knocked out to can not achieve pair
The resistant function of X4 tropics.
The present invention has carried out the knockout detection of CCR5 molecules in immunocyte：By shocking by electricity, rotaring transfecting mode carries gene editing
Body CRISPR/Cas9 and gene targeting carrier pCCR5 donor-LTR-ZFN or pCCR5 donor-MGMTP140K are imported jointly
To primary CD4+In T cell, after five days, screening is carried out by BCNU/BG drugs and is obtained with drug resistant immunocyte；Through streaming
Cell instrument tests and analyzes, the results show that unmodified group (Mock) relatively, transfection has CRISPR/Cas9 and pCCR5 donor-
The CD4 of MGMTP140K+The CD4 of T cell (being named as MGMTP140K) or CRISPR/Cas9 and pCCR5donor-LTR-ZFN+T
CCR5 expression quantity in cell (being named as ZFN) is decreased obviously.
The present invention has carried out genetically modified cell CD4+The antiviral activity detection of T：Virus infection before 24 it is small when, by gene
The CD4 of modification+T cell (MGMTP140K and ZFN) and unmodified CD4+T cell (Mock) is inoculated in 24 orifice plates, Ran Houyong
Genetic modification and unmodified CD4 are infected in centrifugation to the infection of R5 or X4 tropics respectively+T cell, 2 it is small when after, wash away virus
Infection liquid is simultaneously repeated to wash twice with phosphate buffer, and cell, which is resuspended, with complete medium is normally cultivated, three days, six days
Afterwards, detection of expression of the cell conditioned medium for virus protein p24 is collected；P24 detection of expression analysis results are shown, through R5 tropics
After infection, the CD4 of genetic modification+T cell has apparent antivirus action, integrated therein with the cell of antiviral element ZFN
Anti-virus ability is most strong；After the infection of X4 tropics, the CD4 of antiviral element is only integrated with+T cell has antiviral work
With.
In the preparation method of the present invention, the methods of cloned sequence is expanded by PCR modes, DNA sequencing and digestion, is real
It is existing, splice the methods of digestion, annealing, connection cohesive end can be used in sequence and realize.
In the present invention, being applicable in host includes various eucaryotes.
In the present invention, carry for targeting knock out CCR5 genes and destroying the site-directed expression for integrating viral infection resisting
The host cell of the targeting vector of element can be akinete or somatoblast.
The present invention provides a kind of new AntiHIV1 RT activity/AIDS gene targeting carrier and construction method more particularly to structure simultaneously
It obtains the regulation type ZFN Expression elements fixed point insertion HIV-1 infection accessory receptors with targeting excision HIV-1 provirus genes
The targeting vector in CCR5 sites；It is experimentally verified that, human hematopoietic stem cell or immunocyte through the carrier modification are not only due to
The knockout of CCR5 molecules can block the infection of CCR5 preferendum strains, moreover, also can to CXCR4 preferendums strain or the virus leak into,
Provirus gene of the latent infection in host target cells chromosome is cut off, and has more thorough and extensive AntiHIV1 RT activity infection
Activity.
Description of the drawings
Fig. 1 gene targeting carrier schematic diagrames.
Fig. 2 .pCCR5 donor-LTR-ZFN vector gene target practice schematic diagrames.
The orientation restructuring of Fig. 3 genomic levels detection pCCR5 donor-LTR-ZFN carriers.
The bioactivity detection of the antiviral elements of Fig. 4.
The detection of CCR5 developed by molecule amounts in Fig. 5 .TZM-bl cells.
The antiviral activity detection of Fig. 6 genetically modified cells TZM-bl.
Fig. 7 .CD4+The detection of CCR5 developed by molecule amount in T cell.
Fig. 8 genetically modified cells CD4+The antiviral activity detection of T.
Specific embodiment
The genetic recombination detection of 1 gene targeting carrier of embodiment
Using human embryonic kidney cells HEK293 as target cell, by transiently transfecting mode, by gene editing support C RISPR/Cas9
(the two mass ratio is 1 with gene targeting carrier pCCR5 donor-LTR-ZFN：5) cotransfection is into HEK293 cells,
PcDNA3.1 (-) and gene targeting carrier pCCR5 donor-LTR-ZFN cotransfections group are control group；The 5th day after transfection,
Dosing screening is carried out to cell, replaces culture medium for cultivating mdr cell, treats that death no longer occurs in cell, stops dosing sieve
Choosing；Cellular genome is extracted, by integration of the specific primer amplification gene targeting vector in CCR5 sites, to confirm these
The mdr cell survived is the positive cell of genetic modification, and agarose gel electrophoresis testing result is shown, is infected in corotation
In the cell of CRISPR/Cas9 and gene targeting carrier pCCR5 donor-LTR-ZFN, it can detect in gene targeting carrier
The site-directed integration band of upstream and downstream homology arm, size is with being expected unanimously；Only transfecting in pCCR5 donor-LTR-ZFN groups
It finds no purpose band and (as shown in Figure 3) occurs,
It is purpose sequence in order to further determine amplified band, amplified production is purified and is connected in sequencing vector carries out
Verification, the results show that gained band is really expected sequence (as shown in Figure 3), as a result, it was confirmed that constructed gene targeting carrier
Really orientation restructuring can occur at CCR5 sites.
The bioactivity detection of 2 antiviral element of embodiment
The genetically modified cell of above-mentioned acquisition is inoculated in 24 orifice plates, then will carry luciferase reporter gene
The HIV-1 virus particles pHIV-NL4-3-luc and reference gene expression plasmid pRL-SV40pA of luciferase is transfected to above-mentioned
In two kinds of cell lines, after three days, harvest cell and crack, gained supernatant is used for the detection of uciferase activity；Through double fluorescence
Plain enzyme Reporter Assay System analysis result shows, the control cell lines (MGMTP140K) with being only integrated with riddled basins
It compares, in the cell line for being integrated with the antiviral elements of ZFN, luciferase expression amount has nearly 30% decline (as shown in Figure 4)；
Further to verify the antiviral activity of ZFN, infectd with the false type HIV-1 viruses for carrying GFP reporter genes
Two kinds of genetically modified cell systems are stated, metainfective three days, pass through the GFP positive cells in flow cytomery different disposal group
The variation of number, is analyzed through flow cytomery, the results show that compared with cellular control unit system, is integrated with the antiviral members of ZFN
The GFP positive cell numbers infected in the cell line of part have 11% decline, further confirm that ZFN has certain bioactivity
(as shown in Figure 4).
The detection of CCR5 developed by molecule amounts in embodiment 3TZM-bl cells
To express CCR5 with the TZM-bl cells of CXCR4 molecules as target cell, by transiently transfecting mode by gene editing
Support C RISPR/Cas9 and gene targeting carrier pCCR5 donor-MGMTP140K or pCCR5 donor-LTR-ZFN are led jointly
Enter into TZM-bl, the 5th day, screened by BCNU/BG dual drugs, treat that death, the cell of acquisition no longer occurs in cell
Just it is mdr cell；Then culture is enlarged, finally, passes through CCR5 expression quantity in the above-mentioned mdr cell of flow cytomery
Variation；Analyzed through flow cytomery, the results show that opposite untreated fish group (Mock), transfection have gene editing carrier and
CCR5 expression quantity in the TZM-bl cells (being respectively designated as MGMTP140K and ZFN) of gene targeting carrier is decreased obviously (as schemed
Shown in 5).
The antiviral activity detection of 4 genetically modified cell TZM-bl of embodiment
Virus infection the previous day, by TZM-bl cells (MGMTP140K and ZFN) of CCR5 gene knockouts and unmodified
TZM-bl cells (Mock) are inoculated in 24 orifice plates, next day, stood with R5 or X4 tropics infect above-mentioned cell 2 it is small when, it
Removal virus infects liquid and is replaced with complete medium and continues to cultivate afterwards, and metainfective 3rd day of virus is collected in each hole
Cell is simultaneously cracked, and gained supernatant is used for the measure of uciferase activity；Through luciferase reporter gene detecting system
Analysis, the results show that after the infection of R5 tropics, luciferase expression amount intracellular TZM-bl after genetic modification is apparent
Less than unmodified TZM-bl cells, wherein, knocking out integrations in CCR5 has the TZM-bl of antiviral element ZFN intracellular
Luciferase expression amount is significantly lower than the TZM-bl cells for having riddled basins MGMTP140K in CCR5 knockout integrations；
After the infection of X4 tropics, being only integrated with the intracellular luciferase expression amounts of TZM-bl of ZFN reduces (such as Fig. 6 institutes
Show), there are the modified cells of antiviral element ZFN can be to R5 preferendums and X4 preferendums as a result, it was confirmed that knocking out integrations in CCR5
Virus carries out dual resistance；Only knockout CCR5 molecules can not achieve the resistant function to anphotropc virus.
Embodiment 5CD4+The detection of CCR5 developed by molecule amount in T cell
By the rotaring transfecting mode that shocks by electricity by gene editing support C RISPR/Cas9 and gene targeting carrier pCCR5donor-
MGMTP140K or pCCR5donor-LTR-ZFN is directed into primary CD4 jointly+In T cell, after five days, pass through BCNU/BG drugs
It is screened, until obtaining the CD4 with drug resistance+T cell (is respectively designated as MGMTP140K and ZFN), after it is thin through streaming
The variation of CCR5 expression quantity in born of the same parents' instrument detection mdr cell；Through flow detection and analysis, the results show that unmodified group relatively
(Mock), transfection has the CD4 of gene editing carrier and gene targeting carrier+CCR5 tables in T cell (MGMTP140K and ZFN)
It is decreased obviously (as shown in Figure 7) up to amount.
6 genetically modified cell CD4 of embodiment+The antiviral activity detection of T
Virus infection before 24 it is small when, by the CD4 of genetic modification+T cell (MGMTP140K and ZFN) and unmodified CD4+T
Cell (Mock) is inoculated in 24 orifice plates, with R5 or X4 tropics infection centrifugation infection genetic modification and unmodified respectively
CD4+T cell, 2 it is small when after, wash away virus infection liquid and repeat to wash twice with phosphate buffer, be resuspended with complete medium thin
Born of the same parents are normally cultivated；After three days, six days, detection of expression of the cell conditioned medium for virus protein p24 is collected；P24 detection of expression
Analysis result is shown, after the infection of R5 tropics, the CD4 of genetic modification+T cell has apparent antivirus action, wherein
The cell anti-virus ability for being integrated with antiviral element ZFN is most strong；After the infection of X4 tropics, antiviral element is only integrated with
CD4+T cell has antivirus action (as shown in Figure 8).
Claims (7)
1. a kind of gene targeting carrier of AntiHIV1 RT activity/AIDS, which is characterized in that the gene targeting carrier is to pass through targeting knock out
CCR5 molecules are simultaneously carried in the regulation type ZFN expression for destroying site-directed insertion targeting majority HIV-1 virus subtype genes LTR structures
Body, that is, pCCR5 donor-LTR-ZFN plasmids；The gene targeting carrier its be made of following ingredient：
(1) it is used to knock out the upstream and downstream homology arm of CCR5 genes；
(2) the regulation type ZFN Expression elements of virus LTR are targeted；
(3) the riddled basins MGMTP140K Expression elements of enrichment genetically modified cell are beneficial to.
2. the gene targeting carrier of AntiHIV1 RT activity/AIDS according to claim 1, which is characterized in that the gene targeting carrier
In, the pEbackbone of structure is used as recombinant expression carrier.
3. the gene targeting carrier of AntiHIV1 RT activity/AIDS according to claim 1, which is characterized in that the gene targeting carrier
In, entrained ZFN nucleotide fragments are targeting virus LTR in recombinant plasmid pCCR5 donor-LTR-ZFN.
4. the gene targeting carrier of AntiHIV1 RT activity/AIDS according to claim 1, which is characterized in that the gene targeting carrier
In, for being enriched with, the screening-gene MGMTP140K of genetically modified cell is safe in host cell, is sensitively screened.
5. the gene targeting carrier of AntiHIV1 RT activity/AIDS according to claim 4, which is characterized in that the host cell is
Akinete or somatoblast.
6. the gene targeting carrier of AntiHIV1 RT activity/AIDS described in claim 3 is for the HIV infection through the carrier modification
Purposes in human hematopoietic stem cell or immunocyte.
7. the purposes as described in claim 6, which is characterized in that the purposes is, since the knockout of CCR5 molecules can block
The infection of CCR5 preferendum strains and, CXCR4 preferendums strain or the virus leak into, latent infection are dyed in host target cells
Provirus gene in body is cut off.
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