CN106995821A - Jurkat-KI-R5 cell lines and its construction method and application - Google Patents
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Abstract
The present invention relates to a kind of Jurkat-KI-R5 and its construction method and application, strong enhancers/promoters are combined the promoter region that (CAG) knocks in the CCR5 genes of CD4+Jurkat T cells by CRISPR/Cas9 gene editing technology homologous recombinations by the present invention, and the cell line of genetic modification is named as Jurkat-KI-R5 cells.The accurate restructuring of Jurkat-KI-R5 cells CAG promoters of the present invention causes CCR5 stably high expression to CCR5 promoter specified locations.With maternal Jurkat cell on the contrary, Jurkat-KI-R5 cells are easily infected by HIV-1.So, Jurkat-KI-R5 cells provide the cell platform that an AIDS research is badly in need of.
Description
Technical field
The present invention relates to biotechnology, Jurkat-KI-R5 cell lines and its construction method and application are specifically related to.
Background technology
Although antiretroviral therapy prevents and treats AIDS virus, the development in the past few decades is made great effort, AIDS
It is still one of global most important infectious disease.Reported according to UNAIDS, AIDS Global prevalence in 2013,
The estimated quantity of people living with AIDS rises to 35.3 million in 2012 from the 8.1 million of nineteen ninety.In addition, drug resistance
Inhibition of HIV strain has been reported recently.Therefore, the AIDS virus that developing new treatment method is used to remove in patient body is very heavy
The treatment means wanted.The HIV host cell lines of energy infinite multiplication are very important for the development of AIDS research and treatment.
HIV is main using internal macrophage and CD4+T cells as target.In addition, CCR5 and CXCR4 accessory receptors are HIV
Necessary to infection T cell.Therefore, setting up the CD4+T cells of stable expression HIV-1 auxiliary receptor CCR 5s will study non-to HIV
Chang Youyong.However, or existing cell line low expression CCR5 is without supporting R5HIV-1 infection, or it can not be integrated from random
The stable expression CCR5 of transgenosis CCR5 expression vectors into genome.Therefore, a bottleneck problem in HIV researchs is a lack of
Simultaneous Stabilization expresses CD4 and CCR5 T cell system.Even if the CCR5 transgenic T cells system previously set up because expression vector with
Machine is incorporated into genome, and causes the unstable expression of transgenosis.So, set up new mankind's height expression endogenous CCR5 bases
The CD4+T cell lines of cause, play an important role to the new strategy that HIV is treated in research and development.
The content of the invention
Based on this, an object of the present invention provides a kind of method of structure Jurkat-KI-R5 cell lines, and structure is obtained
Jurkat-KI-R5 cell lines can high level expression CCR5.
Realize that the technical scheme of above-mentioned purpose is as follows.
A kind of method for building up of Jurkat-KI-R5 cell lines, comprises the following steps：
A. Jurkat cell is cultivated；
B. pU6-CCR5-gRNA carriers are built：Digestion is carried out to pU6-gRNA carriers with BbsI restriction enzymes, returned
Receive；DNA double chain is inserted, the DNA double chain is annealed into through competent cell turn by SEQ ID NO.2 and SEQ ID NO.3
Change, extract plasmid, obtain pU6-CCR5-gRNA carriers；
C. the structure of recovery template DNA vector, the recovery template DNA vector 5 ' end to 3 ' ends includes CCR5 genes successively
Upstream homology arm, LoxP sites, CAG promoters, resistance screening gene Puro-IRES-Neo, LoxP sites, CAG promoters,
CCR5 downstream of gene homology arms；
D. by the recovery template DNA vector of linearisation, pU6-CCR5-gRNA plasmids and hCas9 plasmid corotation can be expressed
Dye enters Jurkat cell, and nutrient solution is added after transfection and makes cell restoration ecosystem, the culture medium containing puromycin is subsequently added
Monoclonal is isolated in culture, screening, obtains Jurkat-KI-R5 cell lines.
In one of the embodiments, the sequence of the recovery template DNA vector is as shown in SEQ ID NO.4.
In one of the embodiments, the parameter of electrotransfection is：0.4cm electric shock cups, 250volt, 975 μ F, cell quantity
For 5x105-5x106, DNA mass is 1.8-2.2 μ g.
In one of the embodiments, the plasmid that can express hCas9 is pCAG-hCas9 plasmids.
In one of the embodiments, the puromycin concentration in culture medium is 1.5-2.0 μ g/ml.
It is a further object of the present invention to provide a kind of Jurkat-KI-R5 cell lines, it can be used for research and development treating AIDS medicine
The platform of thing.
Realize that the technical scheme of above-mentioned purpose is as follows.
The Jurkat-KI-R5 cell lines obtained according to above-mentioned construction method.
It is a further object of the present invention to provide the application of above-mentioned Jurkat-KI-R5 cell lines.
Application of the above-mentioned Jurkat-KI-R5 cell lines in research and development treating AIDS medicine.
Above-mentioned Jurkat-KI-R5 cell lines as research and development treating AIDS platform application.
To activate the expression of endogenous CCR5 genes, the present invention passes through strong enhancers/promoters combination (CAG)
CRISPR/Cas9 gene editing technology homologous recombinations knock in the promoter region of the CCR5 genes of CD4+Jurkat T cells, base
Because the cell line of modification is named as Jurkat-KI-R5 cells.It is thin in Jurkat-KI-R5T that flow cytometry demonstrates CCR5
The expression of cellular surface.Jurkat-KI-R5 could be the level by detecting the supernatant P24 after HIV by HIV.
The accurate restructuring of Jurkat-KI-R5 cells CAG promoters of the present invention is led to CCR5 promoter specified locations
CCR5 stably high expression is caused, and maternal Jurkat cell is on the contrary, Jurkat-KI-R5 cells are easily infected by HIV-1.So,
Jurkat-KI-R5 cells provide the cell platform that an AIDS research is badly in need of.Using Jurkat-KI-R5 cells as system,
The present invention establishes the technology of the efficient knockout CCR5 genes of CRISPR/CAS9 mediations, is that gene therapy AIDS has established base
Plinth.
Brief description of the drawings
Fig. 1 is the structure of recovery template DNA vector.
Fig. 2 is to utilize CRISPR/CAS9 technologies, and the combination of CAG enhancers/promoters is knocked in into Jurkat by homologous recombination
The promoter site of endogenous cellular CCR5 genes, wherein,
(a) configuration of human endogenous CCR5 gene locis, dark squares representative CCR5 extron, two homology arm Up-Arm and
Dn-Arm is as illustrated, asterisk specifies gRNA target spot；
(b) after homologous recombination allele configuration；
(c) CAG knocks in the PCR identifications of clone.Primer a and b expand 1297bp wild-type fragment, and primer A and B amplification
Allele 750bp fragment after restructuring；
(d) Southern blotting verify the homologous recombination of allele；
(e) CCR5 is in CD4+The height expression of Jurkat-KI-R5 cell surfaces；
(f) Jurkat-KI-R5 cells support HIV-1 infection.
Embodiment
In order to be more clearly understood that the technology contents of the present invention, it is described with reference to the accompanying drawings especially exemplified by following examples.
It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.
The experimental method of unreceipted actual conditions in the following example, generally according to normal condition, such as Sambrook
People, molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) in institute
The condition stated, or according to the condition proposed by manufacturer.Used various conventional chemical reagent, are commercially available in embodiment
Product.
Embodiment 1：The foundation of Jurkat-KI-R5 cells
1.1 cell culture
Jurkat cell (Number:TIB-152TM) culture density maintains 0.5-2x106Cells/mL,
10% heat-inactivated hyclone, 100units/ml penicillin, 100 μ g/mL streptomysins are added in basal medium RPMI1640
Cultivated with 2mmol/L l-glutamines.
1.2DNA vector construction
The DNA vector of encoding humanized Cas9 albumen is purchased from Addgene (article No.s：51142).Produce targeting CCR5 gene positions
The gRNA of point DNA vector pU6-CCR5-gRNA is by pU6-gRNA plasmids (Addgene article No.s：53062) by connecting related target
To the annealed dna double-strand of sequence to two BbsI restriction enzyme sites or other IIS types restriction enzymes (Type IIS
Restriction enzyme) site is built-up.The DNA sites of targeting are GTGCACAGGGTGGAACAAGA.Recovery template
Plasmid both sides have the homology arms of two CCR5 genes, setting up password ATG of its middle and upper reaches homology arm in CCR5 genes respectively
Upstream, ATG and downstream of the downstream homology arm comprising CCR5 genes, and then upstream homology arm is neomycin/puromycin
There is LOXP sites in resistance screening gene, the gene both sides, behind resistance screening gene is to be used to be overexpressed CCR5 genes
CAG promoters.Donor plasmid is analyzed by DNA sequencing and examined.Specific construction step is as follows：
1.2.1pU6-CCR5-gRNA the structure of carrier
Digestion is carried out to pU6-gRNA carriers using BbsI restriction enzymes, be used for after glue reclaim the carrier of next step with
The DNA fragmentation of insertion is attached.The DNA fragmentation of insertion is formed by two single-stranded nucleotides are annealed into DNA double chain, the DNA
Fragment includes targeting sequence and the correlated series being connected with carrier otch, and targeting sequence is GTGCACAGGGTGGAACAAGA (SEQ
ID NO.1), single-stranded DNA sequence 1 (Oligo-F) is 5'-TGCACAGGGTGGAACAAGAGTTTT-3'(SEQ ID NO.2),
Single-stranded DNA sequence 2 (Oligo-R) is 5'-TCTTGTTCCACCCTGTGCACGGTG-3'(SEQ ID NO.3).SEQ ID
NO.2 and SEQ ID NO.3 italicized item reverse complemental.Carried out according to kit specification, the carrier after digestion glue reclaim with
Insertion DNA fragmentation after annealing is attached in the presence of T7 ligases, converted through competent cell, small upgrading grain, digestion
Identification and DNA sequence dna determine that correct pU6-CCR5-gRNA carriers are used for the cell transfection assays of next step after being sequenced.Specifically such as
Under：
In 10ul linked systems, draw 5ul connection products and add the DH5Alpha competent cells that 50ul dissolves on ice,
Gently play it is even, on ice be incubated 30 minutes, subsequent 42 DEG C of water-baths heat shock 30-60s is incubated 2-5 minutes, is subsequently added on ice after heat shock
950ul or so LB culture mediums or recovery 30-60 minutes of 37 DEG C of SOC culture mediums, are evenly coated in the LB- with ampicillin
On Agar Bacteria Culture plates.After bacterial clone is grown, monoclonal is selected, is carried out according to the specification of the small extraction reagent kit of plasmid small
Carry.It is small carry after take 200ng plasmids, with restriction enzyme NheI and NotI (include but is not limited to both restriction enzymes,
Also other can be used any can to identify the enzyme of the plasmid) cut according to specification, choose correct plasmid and carry out DNA surveys
Sequence, determines that plasmid inserts correct sequence.The cell transfecting that correct pU6-CCR5-gRNA carriers are used for into next step is real
Test.
1.2.2 the structure of recovery template carrier
Recovery template DNA vector is built by common molecular clone technology, and particular sequence is as follows：The sequence of italicized item
For CCR5 upstream region of gene homology arms, the sequence of underscore part is LoxP sites, and underscore adds the sequence of italicized item to be opened for CAG
Mover, the sequence of bolded section is resistance screening gene Puro-IRES-Neo, and the sequence of dash area is same for CCR5 downstream of gene
Source arm.Its structure is as shown in Figure 1.Its complete sequence the present embodiment is preferably such as SEQ ID NO.4.Donor plasmid by using
Gibson assembly methods are built and sequence analysis is examined.
SEQ ID NO.4:
GATATCCTGCAGCCCAATTCCGATCATATTCAATAACCCTTAAT TAGGTCTGAAGAGGAGTTTACGT
TGCAGCGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACC
CGCCCGCCCCACGACCCGCAGCGCCCGACCGAAAGGAGCGCACGACCCCATGCATCGTCTAGAGCTCGCTGATCAGC
CTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCA
CTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT
GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGC
TTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGC GGAACCCTTAATTAGGTCCCTCGAGGGCAATTGA T
1.3Jurkat cell transfectings and screening
Linearized using II type gene pulse cell electroporation instrument (Biorad Gene Pulser Xcell) cotransfection
Recovery template plasmid, pU6-CCR5-gRNA plasmids and pCAG-hCas9 plasmids (Addgene companies, plasmid number 51142.And
Other are any to express hCas9 plasmid, including but not limited to other plasmids of Addgene companies and the energy table oneself built
Up to the plasmid of hCas9 albumen) enter Jurkat cell.The design parameter of electrotransfection is：0.4cm electric shock cups, 250volt, 975 μ
F, cell quantity is 1X106, DNA mass is 2 μ g.Nutrient solution is added after transfection and makes cell restoration ecosystem 48 hours, is subsequently added
Containing the culture medium that puromycin concentration is 1.75 μ g/ml, (culture medium is the culture medium of cellar culture Jurkat cell, also may be used
Thinking other any can cultivate the nutrient solution of Jurkat cell) culture 14 days.Divide in the cell survived after puromycin screening
Monoclonal is separated out, 96 orifice plates are seeded in, Jurkat-KI-R5 cells are obtained.
Embodiment 2：Jurkat-KI-R5 cells carry out analysis detection
2.1 flow cytometric analysis
Surface mankind's CCR5 and CD4 albumen is carried out to Jurkat-KI-R5 cells using BD LSR-II flow cytometers
Expression analysis.Data are further analyzed by FACS Diva (Becton Dickinson companies) and FlowJo softwares.1x106
Cell is washed using PBS, at room temperature, is contaminated using DAPI, APC anti-hCCR5 (BD antibody) and PE anti-CD4 (BD antibody)
Color 30 minutes.After dyeing by cells rinsed with PBS twice, and FACS buffer solution is resuspended in, then upper machine analysis.
(e) CCR5 in Fig. 2 is participated in CD4+The height expression of Jurkat-KI-R5 cell surfaces.
The Jurkat-KI-R5CCR5 peaks of left figure APC passages are moved to right it can be seen from Fig. 2 (e), and right figure PE passages
CD4 is overall then without obvious skew.As a result show compared with wild-type Jurkat cells, Jurkat-KI-R5 cell surface CCR5
Expression significantly rise.
2.2PCR detection
The gene knock-in cell line (Jurkat-KI-R5) after homologous recombination is identified using round pcr.Forward primer a (5'-
Tctatgaccttccctgggactt-3', SEQ ID NO.5) and reverse primer d (5'-cttgttccaccctgtgcataa-
3', SEQ ID NO.6) it is used to determine site of the wild-type allele in target gene upstream.Forward primer A (5'-
Tttcgacaccgaagcagagt-3', SEQ ID NO.7) and reverse primer D (5'-gtgcatgttctttgtgggct-3',
SEQ ID NO.8) it is used to identify the downstream part of target gene.On the gene knock-in target cell system after homologous recombination
PCR identify, using with identical forward primer in wild type identification, redesign in addition correlation reverse primer.Reverse primer b
(5'-tcgaacgtaaactcctcttcagac-3'SEQ ID NO.9) is used to verify the upstream arm after homologous recombination, and reverse
Primer B (5'-cgtgctggttattgtgctgtc-3'SEQ ID NO.10) is used to identify the downstream arm after homologous recombination.Referring to
The position of probe and the size of SacI restriction fragments are illustrated in (b) in Fig. 2, figure.PCR primer (a, d, A and D) is used to
Determine wild-type allele.Primer (a, b, A and B) is used to screen the allele knocked in；The size of PCR amplicons is as schemed
Show.PCR amplification programs are：1.94 DEG C 4 minutes；2.94 DEG C 45 seconds；3.57 DEG C 30 seconds；4.72 DEG C repeat to the 2nd step for 45 seconds
39 circulations；5.72 DEG C 5 minutes；6. maintain 4 DEG C.
CAG knocks in the PCR identifications of clone.
Amplification refers to (c) in Fig. 2, and primer A and B expand 1297bp wild-type fragment, and primer A and B expand
Allele 750bp fragment after weightening group.
The primer that Fig. 2 (c) the picture left above is used is a+b, and only Jurkat- is known that according to primer location figure in Fig. 2 (b)
The upstream arm that KI-R5 can amplify 1297bp knocks in allele.The primer that Fig. 2 (c) top right plots are used is a+d, by Fig. 2 (a)
It is known that the cell containing wild-type allele can amplify 1072bp wild-type fragment.Fig. 2 (c) lower-lefts figure is used
Primer be A+B, be known that only Jurkat-KI-R5 can amplify 750bp downstream according to primer location figure in Fig. 2 (b)
Arm knocks in allele.The primer that Fig. 2 (c) bottom-right graphs are used is A+D, by Fig. 2 (a) it is known that containing wild-type allele
Cell can amplify 1368bp wild-type fragment.In summary, Jurkat-KI-R5 belongs to heterozygosis subtype and knocks in cell,
One allele is knocks in type, and another allele is wild type.
2.3Southern blotting methods are analyzed
The genomic DNA of the Jurkat-KI-R5 cells after wild-type Jurkat cells and genetic recombination is extracted respectively, respectively
15 μ g in DNA sample are taken to be digested with restriction enzyme SacI.Postdigestive DNA is separated with 1.0% Ago-Gel, separation
Condition is：30-35V, 160mA, 16 hour.Then by capillarity absorption transferring film, the DNA fragmentation after separation is shifted to nitre
On acid cellulose film, use32The DNA probe of P marks goes to mark CCR5 genes, and whether checking gene is accurately knocked in.Referring in Fig. 2
(d), Jurkat-KI-R5 containing wild type (WT) and knocks in type (KI) specific band simultaneously, shows that gene has knocked in it
In an allele, it is consistent with the PCR amplifications in Fig. 2 (c), Jurkat-KI-R5 be heterozygosis subtype knock in cell.
Downstream (Dn) probe sequence is：atgcctttgtcggggagaagttcagaaactacctcttagtcttcttccaa
aagcacattgccaaacgcttctgcaaatgctgttctattttccagcaagaggctcccgagcgagcaagctcagttta
cacccgatccactggggagcaggaaatatctgtgggcttgtgacacggactcaagtgggctggtgacccagtcagag
ttgtgcacatggcttagttttcatacacagcctgggctgggg gtggggtgggagaggtcttttttaaaaggaagtt
actgttatagagggtctaagattcatccatttatttggcatctgtttaaagtagattagatcttttaagcccatcaa
ttatagaaagccaaatcaaaatatgttgatgaaaaatagcaacctttttatctccccttcacatgcatcaagttatt
gacaaactctcccttcactccgaaagttccttatgtatatttaaaagaaagcctcagagaattgctgattcttgagt
ttagtgatctgaacagaaataccaaaattatttcagaaatgtacaactttttacctagtacaaggcaacatataggt
tgtaaatgtgtttaaaacaggtctttgtcttgctatggggagaaaagacatgaatatgattagtaaagaaatgacac
ttttcatgtgtgatttcccctccaaggtatggttaataagtttcactgacttagaaccaggcgagagacttgtg
(SEQ ID NO.11)。
Internal (In) probe sequence is：gccacaaccatgaccgagtacaagcccacggtgcgcctcgccacccgcga
cgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccgg
accgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgg
gtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagat
cggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcacc
ggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgcc
gtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacct
ccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatga
cccgcaagcccggtgcctgacgcccgccccacgacccgcagcgcccgaccgaaaggagcgcacgaccccatgcatcg
tctagagctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttc
cttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggt
gtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggg
gatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctcgactagagcttgcggaaccct(SEQ
ID NO.12)。
2.4 HIV infections are detected
Wild-type Jurkat cells and Jurkat-CCR5 cells 2 hours are infected using 0.3MOI BaL HIV-1.It is positive right
According to the MOLT-4 cells to be infected using same method, negative control is not infected MOLT-4 cells.The 3rd day after infection
Supernatant was collected with the 7th day, and tests P24 concentration level.All tests are in triplicate.Data statistic analysis be by
The processing of Prism6 programs.Referring to (f) in Fig. 2.The 3rd day infected in HIV-1, negative control MOLT4-no virus do not have inspection
P24 is measured, positive control MOLT4-virus detects the p24 of high concentration, show experimental design rationally, meanwhile, wild type
Jurkat is also not detected by p24, and Jurkat-KI-R5 p24 concentration levels are suitable with positive control, have with wild type Jurkat
Significant difference (p=0.0002)；The 7th day infected in HIV-1, negative control MOLT4-no virus do not detect p24,
Positive control MOLT4-virus detects the p24 of high concentration, shows experimental design rationally, meanwhile, wild type Jurkat is not also examined
P24 is measured, Jurkat-KI-R5 p24 concentration levels are suitable with positive control, have significant difference (p with wild type Jurkat
=0.0001)；As a result show that HIV-1 only infects Jurkat-KI-R5 cells, it is impossible to infect wild-type Jurkat cells.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope of this specification record is all considered to be.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that coming for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (8)
1. a kind of construction method of Jurkat-KI-R5 cell lines, it is characterised in that comprise the following steps：
A. the Jurkat cell in being cultivated；
B. pU6-CCR5-gRNA carriers are built：Digestion is carried out to pU6-gRNA carriers with BbsI restriction enzymes, is reclaimed；Insert
Enter DNA double chain, the DNA double chain is formed by SEQ ID NO.2 and SEQ ID NO.3 are annealed；Convert, carry through competent cell
Plasmid is taken, pU6-CCR5-gRNA carriers are obtained；
C. recovery template DNA vector is built, it is same that the recovery template DNA vector 5 ' end to 3 ' ends includes CCR5 upstream region of gene successively
Source arm, LoxP sites, CAG promoters, resistance screening gene Puro-IRES-Neo, LoxP sites, CAG promoters, CCR5 genes
Downstream homology arm；
D. by the recovery template DNA vector of linearisation, pU6-CCR5-gRNA plasmids and hCas9 plasmid co-transfection can be expressed enter
Enter in Jurkat cell, nutrient solution is added after transfection and makes cell restoration ecosystem, is subsequently added in the culture medium containing puromycin
Monoclonal is isolated in culture, screening, obtains Jurkat-KI-R5 cell lines.
2. the construction method of Jurkat-KI-R5 cell lines according to claim 1, it is characterised in that the recovery template
The sequence of DNA vector is as shown in SEQ ID NO.4.
3. the construction method of Jurkat-KI-R5 cell lines according to claim 1, it is characterised in that fast in culture medium
Purine mycin concentration is 1.5-2.0 μ g/ml.
4. the construction method of Jurkat-KI-R5 cell lines according to claim 1, it is characterised in that the parameter of electrotransfection
For：0.4cm electric shock cups, 250volt, 975 μ F, cell quantity is 5x105-5x106, DNA mass is 1.8-2.2 μ g.
5. the construction method of the Jurkat-KI-R5 cell lines according to claim any one of 1-4, it is characterised in that described
The plasmid that hCas9 can be expressed is pCAG-hCas9 plasmids.
6. the Jurkat-KI-R5 cell lines obtained according to any one of the claim 1-5 construction methods.
7. application of the Jurkat-KI-R5 cell lines described in claim 6 in research and development treating AIDS medicine.
8. Jurkat-KI-R5 cell lines described in claim 6 are used as the application of the platform of research and development treating AIDS.
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