CN107974460A - Chimeric antigen receptor gene for HIV-1, plasmid, T cell, kit and application with the gene - Google Patents
Chimeric antigen receptor gene for HIV-1, plasmid, T cell, kit and application with the gene Download PDFInfo
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Abstract
The invention discloses a kind of Chimeric antigen receptor gene for HIV 1, the gene includes guiding sub-district, the domain combined with the CD4 binding sites of gp120, hinge area, transmembrane domain, costimulation domain, signal transduction domain and suicide gene system area, and the present invention discloses the plasmid with the gene, T cell, kit and application.T cell provided by the invention is in use, the infection of HIV 1 can be prevented, while increase the lethality to 1 infection cells of HIV.When adverse reaction occurs after inputting the 1 CAR T cells of HIV with the present invention, internal cell can be removed by suicide gene system at once, to reach prevention effect.
Description
Technical field
The present invention relates to gene technology field, more particularly to a kind of Chimeric antigen receptor gene for HIV-1, have should
Plasmid, T cell, kit and the application of gene.
Background technology
Human immunodeficiency virus (human immunodeficiency virus Type 1, HIV-1), belongs to reverse transcription
One kind of virus, invades CD4+T cells, body acquired immunity function is badly damaged, ultimately result in AIDS AIDS
(Acquired immunodeficiency syndrome).The Chinese Center for Disease Control and Prevention of in August, 2017 discloses the whole nation
AIDS Epidemic, the number of the infected of the domestic HIV-1 in China (human immunodeficiency virus) have reached 718270
People, wherein thering are 299169 people to suffer from AIDS, because the number of AIDS death has reached 221628 people.In addition HIV- is increased newly every year
1 the infected/patient AIDS is up to more than 140000 people.These numerals show the HIV infection situation very severe in China.
At present, AIDS clinical treatment is mainly highly active antiretroviral therapy (Highly active
Antiretroviral therapy, HAART), which not only effectively controls HIV-1 to replicate, and can rebuild AIDS patient
Immune (Michaels, S.H.；Clark,R.；Kissinger,P.Declining morbidity and mortality
among patients with advanced human immunodeficiency virus
infection.N.Engl.J.Med,1998.339:405–406.).However, due to viral integrase in infected cell and shape
The latent infection repository stable into one, patient once stop drug therapy, and virus load is horizontal can rebound to treatment again before
(David,D.Ho.Toward HIV-1eradication or remission:the tasks ahead.Science,
1998.280:1866-1867.).Therefore, which can not thoroughly remove the HIV-1 of patient's body.In order to keep drug effect, patient
Must perennially it take medicine, not only costly (needing ten thousand dollars of 1.2-5.0 for each person every year), it is secondary also to produce serious poison
(infringement kidney and liver, cause heart disease, and diabetes, weaken bone, cause weight gain and mental disease problem, such as lose for effect
Dormancy and depression etc.).In view of the above problems, the usual developmental function of the prior art is in novel targets and has the efficient of new construction type
Low toxicity, HIV-1 inhibitor (United States Patent (USP) 5,849,911,6,166,004 and 6,087,383 etc. of overriding resistance；Chinese patent
CN200480010780.2 and CN03814963.X etc.).But HIV-1 genes have variability, in HIV-1 inhibitor medicines
Easily undergoing mutation under thing selection pressure causes drug resistance strain to produce, so as to reduce the effective of HIV-1 inhibitor medicaments treatment
Property.And existing technical solution does not allow patient thoroughly to break away from the situation of long-term administration.Therefore, people are badly in need of finding treatment
The new method of HIV-1 infection.
The cytolysis patent of HIV-1 infection cells is directed in the cell by carrying chimeric CD4 acceptors
All disclosed in CN95192559.8 and cell and correlation molecule and process patent CN95195183.1 with CD4 decoy acceptors
For the T cell therapy of HIV-1.The therapy shows in the case of HAART treatments are not carried out, by the T cell of genetic modification
HIV-1 viruses can be efficiently controlled.Its mechanism of action is mainly, by CD4 extracellular domains and intracellular CD3- ζ signal structures
The be connected CD4- ζ Chimeric antigen receptors (chimeric antigen receptor, CAR) of composition of domain are transfected by viral vector
To human T-cell, since CD4 is the major receptors of HIV-1 infection cells, the CD4 extracellular domains of CD4- ζ CAR pass through bound site
Immunological synapse is simulated in the HIV-1 outer membrane proteins gp120 on infection cell surface, causes CD4- ζ CAR-T cell activations, and then
HIV-1 infection cells are cracked, reduce the HIV-1 virus loads of patient's body.But CD4- ζ CAR-T cells are with reference to gp120
After antigen, although the T cell that can become activation kills the HIV-1 infection cells combined, the T cell of activation is difficult into one
Step propagation can only play temporal effect, and CD4- ζ CAR-T cells are easily hiv-1-infected, become new host.Therefore in the later stage
Clinical trial result is unsatisfactory, so far, is not widely used also.
Therefore, a kind of new safe efficient type Chimeric antigen receptor for HIV-1 is developed, not only there is urgent grind
Study carefully value, it may have good economic benefit and extensive medical applications potentiality, this is exactly the power institute that the present invention is accomplished
And basis.
The content of the invention
The defects of in order to overcome the prior art as indicated above, the present inventor has made intensive studies this, is paying
After a large amount of creative works, so as to complete the present invention.
Specifically, the technical problems to be solved by the invention are：There is provided for HIV-1 Chimeric antigen receptor gene,
Plasmid, T cell, kit and application with the gene.
In order to solve the above technical problems, the technical scheme is that：
In a first aspect, the present invention provides the Chimeric antigen receptor gene for HIV-1, the gene include guiding sub-district,
The domain that is combined with the CD4 binding sites of gp120, hinge area, transmembrane domain, costimulation domain, signal transduction structure
Domain and suicide gene system area.
In the present invention, as a kind of perferred technical scheme, the guiding sub-district guides sub-district for CD8.
In the present invention, as a kind of perferred technical scheme, the domain combined with the CD4 binding sites of gp120
Using any one in T4 antigen, VRC01, VRC01b, VRC01-C, 3BNC60, NIH45-46.
In the present invention, as a kind of perferred technical scheme, the domain combined with the CD4 binding sites of gp120
Using T4 antigen.
In the present invention, as a kind of perferred technical scheme, the hinge area of the Chimeric antigen receptor is CD8 hinge areas.
In the present invention, as a kind of perferred technical scheme, the transmembrane domain of the Chimeric antigen receptor is CD8 cross-films
Area.
In the present invention, as a kind of perferred technical scheme, the costimulation domain of the Chimeric antigen receptor is 4-1BB
Functional domain.
In the present invention, as a kind of perferred technical scheme, the signal transduction domain of the Chimeric antigen receptor is CD3
The functional domain of ζ.
In the present invention, as a kind of perferred technical scheme, the suicide gene system of the Chimeric antigen receptor is
ICasp9 suicide gene systems.
It is corresponding, nucleotide sequence such as SEQ ID NO.1, the SEQ of the Chimeric antigen receptor gene for HIV-1
ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14。
In the present invention, as a kind of perferred technical scheme, the Chimeric antigen receptor gene for HIV-1 is preferably
Following block coupled in series obtains：
CD8 guides sub-district nucleic acid artificial sequence, such as SEQ ID NO.2；
T4 antigen nucleic acid artificial sequence, such as SEQ ID NO.3；
CD8 hinge area nucleic acid artificial sequences, such as SEQ ID NO.4；
CD8 transmembrane region nucleic acid artificial sequences, such as SEQ ID NO.5；
4-1BB costimulations area nucleic acid artificial sequence, such as SEQ ID NO.6；
CD3 ζ signal transductions area nucleic acid artificial sequence, such as SEQ ID NO.7；
Autothermic cracking peptide T 2A nucleic acid artificial sequences, such as SEQ ID NO.8；
ICasp9 suicide genes area nucleic acid artificial sequence, SEQ ID NO.9.
Obtained for the Chimeric antigen receptor gene of HIV-1 using the preparation method included the following steps：
(1) respectively by CD8 guiding sub-district nucleic acid artificial sequence, T4 antigen nucleic acid artificial sequence, CD8 hinge area nucleic acid people
Process row, CD8 transmembrane region nucleic acid artificial sequence, 4-1BB costimulations area nucleic acid artificial sequence, CD3 ζ signal transductions area nucleic acid are artificial
Sequence, autothermic cracking peptide T 2A nucleic acid artificial sequence, iCasp9 suicide genes area nucleic acid artificial sequence synthesize its whole expression cassette simultaneously
PUC-HIV-1-CD4-CAR is obtained on insertion standard vector pUC57；
(2) pUC-HIV-1-CD4-CAR is subjected to double digestion, will contains HIV-1-CD4-CAR DNA using agar-agar electrophoresis
Fragment agar-agar position is cut, handled using DNA extraction kit sol solutions, cross DF columns abandon filtrate, rinsing DF columns, it is empty from,
Elute DF columns, collect centrifugation thing, the HIV-1-CD4-CAR DNA fragmentations purified are that is, of the present invention for HIV-1's
Chimeric antigen receptor gene.
Second aspect, the present invention provides a kind of plasmid, the plasmid includes the inosculating antibody as described above for being directed to HIV-1
Original receptor gene.
In the present invention, as a kind of perferred technical scheme, the plasmid is by fusion fragment CD8leader-
CD4-CD8-4-1BB-CD3 ζ-T2A-iCasp9 insertions Lentiviral pLent-C-GFP is obtained.
The plasmid is prepared using following preparation method：By the HIV-1-CD4-CAR DNA fragmentations and line of above-mentioned purifying
The pLent-C-GFP DNA fragmentations of property, are in linked system：10×buffer：1μl；T4 ligases：1μl；HIV-1-CD4-
CAR DNA：4μl；The pLent-C-GFP DNA of linearisation：Connection forms pLent-HIV-1-CD4- overnight under conditions of 4 μ l
CAR plasmids.
The third aspect, the present invention provides T cell, the T cell includes the chimeric antigen as described above for being directed to HIV-1
Acceptor gene.
In the present invention, as a kind of perferred technical scheme, the T cell is prepared using following preparation method：It is first
Above-mentioned pLent-HIV-1-CD4-CAR plasmids are first subjected to slow virus packaging, then infect T cell using recombinant slow virus.
Fourth aspect, the present invention provides a kind of kit, including
Obtain the carrier as described above for stablizing expression HIV-1-CD4-CAR；
And carrier diluent.
5th aspect, the present invention provides the application of the gene, refers to gene energy in the medicine for preparing treatment HIV-1
Enough applied.The form of the medicine includes but not limited to kit.
After employing above-mentioned technical proposal, the beneficial effects of the invention are as follows：
The present invention structure that the CD4 binding sites with gp120 are combined is combined with CAR motifs, respectively as AntiHIV1 RT activity-
The films of 1CAR molecules is outer and film intracellular domain, and the design to CAR is transformed, thus develop it is a kind of completely newly, MHC it is non-
The anti-HIV-1 CAR molecules of dependence, this patent are named as HIV-1-CAR (see Fig. 1).HIV-1-CAR, on the one hand, reduce HIV
The homology of cell receptor, makes HIV-1-CAR-T cells from HIV infection；On the other hand, HIV-1-CAR can also be further
Amplifier T cell activation signal, improves internal amplification ability and produces cell factor generation ability.HIV-1-CAR-T and expression
The cell line row of gp120 co-cultures, and can forcefully kill the cell line of expression gp120.And fragmentation effect is substantially better than
Compared to the CD4- ζ CAR-T cells reported in the early time.For more detailed, the present invention is using CD8 guiding sub-districts and hinge area, in reality
The homology of CD4 can be reduced by trampling middle confirmation, prevent the CAR-T of modification from being infected by HIV-1.Can further it be swashed using 4-1BB
The CAR-T of modification living, makes CAR-T largely expand, increases the lethality to HIV-1 infection cells.
The HIV-1-CAR-T of the present invention carries iCasp9 suicide gene systems, once HIV-1- of the input with the present invention
When adverse reaction occurring after CAR-T cells, internal cell can be removed by suicide gene system at once, be made with reaching prevention
With.
Brief description of the drawings
Fig. 1 is safe efficient type Chimeric antigen receptor principle design drawing of the present invention.
Fig. 2 is HIV-1-CD4-CAR DNA fragmentations (right side) and pLent-C-GFP DNA (left side) piece of linearisation in the present invention
Section electrophoretogram.
Fig. 3 expresses matter for slow virus CD8leader-CD4-CD8-4-1BB-CD3 ζ-T2A-iCasp9 of the present invention
The schematic diagram of grain.
The efficiency that Fig. 4 is the expression HIV-1-CD4-CAR of HIV-1-CD4-CAR-T cells of the present invention is 81.8%.
Fig. 5 is ELIspot of the present invention experiment detection figures, and left figure is the situation of HIV-1-CD4-CAR-T secretion of gamma-IFN；In
Figure is the situation of CD4- ζ CAR-T secretion of gamma-IFN；Right figure is not transform the situation of CD8+T cell secretion of gamma-IFN；Express HIV-
The secretion capacity of the HIV-1-CD4-CAR-T cell IFN-γ of 1NL4-3 envelope glycoprotein cell lines Jurkat mixed culture is notable
CD8+T cells are not transformed higher than CD4- ζ CAR-T and.
Fig. 6 is the killing experiments result figure of HIV-1-CD4-CAR-T cells of the present invention.In E：T is 1：When 1, HIV-1-
CD4-CAR-T is obvious for 90.56 ± 8.23% to the killing-efficiency for expressing HIV-1NL4-3 envelope glycoprotein cell lines Jurkat
CD8+T cells are not transformed higher than CD4- ζ CAR-T and；
Fig. 7 is the killing experiments result figure of HIV-1-CAR-T cells of the present invention.In E：T is 1：When 1, HIV-1-CAR-T pairs
The killing-efficiency of HIV-1NL4-3 envelope glycoprotein cell lines Jurkat is expressed up to more than 85%, hence it is evident that higher than CD4- ζ CAR-T
CD8+T cells are not transformed.
Embodiment
With reference to specific embodiment, the present invention is further described.But the purposes and mesh of these exemplary embodiments
Be only used for enumerate the present invention, any type of any restriction not is formed to the real protection scope of the present invention, it is more non-to incite somebody to action this
The protection domain of invention is confined to this.
Embodiment 1
For the Chimeric antigen receptor gene of HIV-1, which includes guiding sub-district, the CD4 binding site knots with gp120
Domain, hinge area, transmembrane domain, costimulation domain, signal transduction domain and the suicide gene system area of conjunction.Its
In, the domain combined with the CD4 binding sites of gp120 using T4 antigen, VRC01, VRC01b, VRC01-C,
Any one in 3BNC60, NIH45-46.
The present embodiment as a kind of preferable scheme, for HIV-1 Chimeric antigen receptor gene nucleotide sequence such as
SEQ ID NO.1.I.e.：Obtained for following block coupled in series：CD8 guides sub-district nucleic acid artificial sequence, such as SEQ ID NO.2；CD4 resists
Protokaryon acid artificial sequence, such as SEQ ID NO.3；CD8 hinge area nucleic acid artificial sequences, such as SEQ ID NO.4；CD8 transmembrane region cores
Sour artificial sequence, such as SEQ ID NO.5；4-1BB costimulations area nucleic acid artificial sequence, such as SEQ ID NO.6；CD3 ζ signal transductions
Area's nucleic acid artificial sequence, such as SEQ ID NO.7；Autothermic cracking peptide T 2A nucleic acid artificial sequences, such as SEQ ID NO.8；ICasp9 is certainly
Gene regions nucleic acid artificial sequence is killed, such as SEQ ID NO.9.
Embodiment 2
For the preparation embodiment of the Chimeric antigen receptor gene of HIV-1.
The present embodiment is also to be said so that the domain that the CD4 binding sites with gp120 are combined uses T4 antigen as an example
It is bright, when using VRC01 (SEQ ID NO.10), VRC01b (SEQ ID NO.11), VRC01-C (SEQ ID NO.12),
In 3BNC60 (SEQ ID NO.13), NIH45-46 (SEQ ID NO.14) it is other any one when, the basic phase of its preparation method
Together, do not do and excessively repeat herein.
For the preparation method of the Chimeric antigen receptor gene of HIV-1, include the following steps：
(1) respectively by CD8 guiding sub-district nucleic acid artificial sequence, T4 antigen nucleic acid artificial sequence, CD8 hinge area nucleic acid people
Process row, CD8 transmembrane region nucleic acid artificial sequence, 4-1BB costimulations area nucleic acid artificial sequence, CD3 ζ signal transductions area nucleic acid are artificial
Sequence, autothermic cracking peptide T 2A nucleic acid artificial sequence, iCasp9 suicide genes area nucleic acid artificial sequence synthesize its whole expression cassette simultaneously
PUC-HIV-1-CD4-CAR is obtained on insertion standard vector pUC57；
(2) pUC-HIV-1-CD4-CAR is subjected to double digestion, will contains HIV-1-CD4-CAR DNA using agar-agar electrophoresis
Fragment agar-agar position is cut, handled using DNA extraction kit sol solutions, cross DF columns abandon filtrate, rinsing DF columns, it is empty from,
Elute DF columns, collect centrifugation thing, the HIV-1-CD4-CAR DNA fragmentations purified are that is, of the present invention for HIV-1's
Chimeric antigen receptor gene.
For more detailed, the preparation method of the Chimeric antigen receptor gene provided in this embodiment for HIV-1, including
Following steps：
Respectively by CD8 guiding sub-district nucleic acid artificial sequence, T4 antigen nucleic acid artificial sequence, CD8 hinge area nucleic acid people's processes
Row, CD8 transmembrane region nucleic acid artificial sequence, 4-1BB costimulations area nucleic acid artificial sequence, CD3 ζ signal transductions area nucleic acid people's process
Row, autothermic cracking peptide T 2A nucleic acid artificial sequence, iCasp9 suicide genes area nucleic acid artificial sequence student on commission's work bioengineering (on
Sea) Co., Ltd synthesizes its whole expression cassette and is inserted on standard vector pUC57, therefore is named as pUC-HIV-1-CD4-CAR,
PUC-HIV-1-CD4-CAR is subjected to Fast Digest AsiSI (being purchased from ThermoFisher companies) and Fast
Digest NotI (being purchased from ThermoFisher companies) double digestion, 37 DEG C, digestion 20min.100 μ l digestion systems are：10×
buffer：10μl；DNA 6μg；AsiSI enzymes：3μl；NotI enzymes：3μl；Deionized water supplies volume.
The agar-agar position containing HIV-1-CD4-CAR DNA fragmentations will be cut using agar-agar electrophoresis, be placed on two centrifugations
Guan Zhong.DNA dissolution and is concentrated from agar-agar using DNA extraction kit (being purchased from ThermoFisher companies), first
500 μ l DF buffer are added toward above-mentioned centrifuge tube, 55 DEG C act on 10 minutes, per rocking once within 2-3 minutes, until agar-agar is complete
Dissolving.Again by agar-agar solution all suction DF Column, and put on Collection Tube (collection filtered fluid).8000rpm
Centrifugation 1 minute, filtered fluid is outwelled.500 μ l Wash Buffer, 8000rpm centrifugation 1 minute is added, filtered fluid is outwelled.
12000rpm, which is centrifuged 2 minutes, ensures that ethanol is removed.DF Column are finally transferred to upper another clean microcentrifugal tube,
25 μ l Elution Buffer are added, after being stored at room temperature 2 minutes, 14000rpm is centrifuged 2 minutes, the liquid in microcentrifugal tube
The HIV-1-CD4-CAR DNA fragmentations as purified (see Fig. 2).
Embodiment 3
A kind of plasmid, the plasmid include the Chimeric antigen receptor gene as described above for being directed to HIV-1.The present embodiment
Plasmid is that fusion fragment CD8leader-CD4-CD8-4-1BB-CD3 ζ-T2A-iCasp9 are inserted into Lentiviral
PLent-C-GFP is obtained.
Embodiment 4
The preparation embodiment of plasmid.
Plasmid is prepared using following preparation method：By the HIV-1-CD4-CAR DNA fragmentations of above-mentioned purifying and linearly
The pLent-C-GFP DNA fragmentations of change, are in linked system：10×buffer：1μl；T4 ligases：1μl；HIV-1-CD4-
CAR DNA：4μl；The pLent-C-GFP DNA of linearisation：Connection forms pLent-HIV-1-CD4- overnight under conditions of 4 μ l
CAR plasmids.
In more detail, the process for preparing plasmid that the present embodiment uses, includes the following steps：
Respectively by CD8 guiding sub-district nucleic acid artificial sequence, T4 antigen nucleic acid artificial sequence, CD8 hinge area nucleic acid people's processes
Row, CD8 transmembrane region nucleic acid artificial sequence, 4-1BB costimulations area nucleic acid artificial sequence, CD3 ζ signal transductions area nucleic acid people's process
Row, autothermic cracking peptide T 2A nucleic acid artificial sequence, iCasp9 suicide genes area nucleic acid artificial sequence student on commission's work bioengineering (on
Sea) Co., Ltd synthesizes its whole expression cassette and is inserted on standard vector pUC, therefore is named as pUC-HIV-1-CD4-CAR, together
When by pUC-HIV-1-CD4-CAR and pLent-C-GFP carriers progress Fast Digest AsiSI (be purchased from ThermoFisher
Company) and Fast Digest NotI (being purchased from ThermoFisher companies) double digestion, 37 DEG C, digestion 20min.100 μ l digestions
System is：10×buffer：10μl；DNA 6μg；AsiSI enzymes：3μl；NotI enzymes：3μl；Deionized water supplies volume.Utilize fine jade
Gel electrophoresis are by respectively the agar-agar portion of the pLent-C-GFP DNA fragmentations containing HIV-1-CD4-CAR DNA fragmentations and linearisation
Position is cut, and is placed in two centrifuge tubes.Using DNA extraction kit (being purchased from ThermoFisher companies) by DNA from fine jade
Dissolution and concentrated in glue, add 500 μ l DF buffer toward above-mentioned centrifuge tube first, 55 DEG C act on 10 minutes, per shaking within 2-3 minutes
Shake once, until agar-agar is completely dissolved.Again by agar-agar solution all suction DF Column, and put on Collection Tube
(collection filtered fluid).8000rpm is centrifuged 1 minute, and filtered fluid is outwelled.Add 500 μ l Wash Buffer, 8000rpm from
The heart 1 minute, filtered fluid is outwelled.12000rpm, which is centrifuged 2 minutes, ensures that ethanol is removed.Finally DF Column are transferred to another
Clean microcentrifugal tube, adds 25 μ l Elution Buffer, and after being stored at room temperature 2 minutes, 14000rpm is centrifuged 2 minutes, micro-
Measure the pLent-C-GFP that the liquid in centrifuge tube is HIV-1-CD4-CAR DNA fragmentations (see Fig. 2) and the linearisation purified
DNA (see Fig. 2) fragment.
Above two DNA fragmentation is subjected to overnight connection at 16 DEG C and forms pLent-HIV-1-CD4-CAR (see Fig. 3) matter
Grain.Linked system is：10×buffer：1μl；T4 ligases：1μl；HIV-1-CD4-CAR DNA：4μl；Linearisation
pLent-C-GFP DNA：4μl.
Embodiment 5
The purifying embodiment of pLent-HIV-1-CD4-CAR plasmids.
Above-mentioned pLent-HIV-1-CD4-CAR is transformed into E.coli (DH5 α).Comprise the following steps that：By plasmid and impression
State cell mixes and is incubated half an hour on ice, then 42 degree heat shocks 90 seconds, then places 2min on ice, finally adds LB liquid medium to delay
Shaking or so 1 hour, 3000rpm centrifuges 5min again, and 100 μ l bacterium solutions are coated on containing ammonia benzyl LB solid plates.
Next day picking single bacterium colony is incubated overnight, and is extracted using plasmid extraction purification kit (being purchased from Qiagen companies)
PLent-HIV-1-CD4-CAR plasmids, comprise the following steps that：(1) 10000 × g of 1.5ml bacterium solution room temperatures is taken to centrifuge 1min.(2) go
Supernatant, adds 250 μ l solution I (A containing RNase), and vortex oscillator is shaken to thalline to suspend completely.(3) 250 μ l solution II are added,
Gently overturn centrifuge tube 4~6 times, obtain the lysate of clarification.Preferably it is incubated at room temperature 2min.(4) 350 μ l solution III are added, gently
Reverse to mix for several times, to there is white flock precipitate, 10000 × g of room temperature centrifuges 10min.(5) especially careful absorption supernatant, is moved
Into the clean absorbing column for assembling volume 2ml centrifuge tubes.Ensure not suck precipitation and cell fragment.Room temperature 10000
× g centrifuges 1min, passes through absorbing column completely to lysate.(6) abandon filtered solution, add 500 μ l Buffer HBC, 10000 × g from
Heart 1min, cleans absorbing column, removes the purity that residual protein ensures DNA.(7) filtered solution is abandoned, then it is diluted with 100% ethanol
750 μ l Wash Buffer clean absorbing column, 10000 × g centrifugations 1min.(8) 750 μ l Wash Buffer cleanings are added to absorb again
Column.(9) 10000 × g of absorbing column must be centrifuged 2min ensures that ethanol is removed.(10) by absorbing column be put into clean 1.5ml from
Heart pipe, adds 50-100 μ l the final concentration of needs (depend on) aseptic deionized water or TE buffer solutions on filter membrane, 10000 × g from
Heart 5min, collects Plasmid DNA.(11) and precognition concentration DNA sample (Marker) does agarose gel electrophoresis, contrast knot together
Fruit, it is 332ng/ μ l to draw pLent-HIV-1-CD4-CAR plasmid concentrations.
Sangon Biotech (Shanghai) Co., Ltd. is entrusted to be surveyed above-mentioned pLent-HIV-1-CD4-CAR plasmids
Sequence.It is spare after being sequenced correctly.
Embodiment 6
T cell, the T cell include the Chimeric antigen receptor gene as described above for being directed to HIV-1.The T of the present embodiment
Cell is prepared using following preparation method：Above-mentioned pLent-HIV-1-CD4-CAR plasmids are subjected to slow virus bag first
Dress, then infects T cell using recombinant slow virus.
Following embodiments are slow virus packaging, the preparation of PBMC cells, the method for recombinant slow virus infection T cell.
Embodiment 7
Slow virus packs embodiment.
It is as follows using Lentiviral Packaging Kit slow virus package kits, specific method：By slow virus bag
Dress cell line 293T is inoculated in containing in DMEM+10%FBS 10cm culture dishes, 37 DEG C, is cultivated under the conditions of 5% CO2, adherent
Prepare transfection when rate is 70%-80%.Sterile 1.5ml EP pipes or 15ml centrifuge tubes are taken, reactant is prepared by following component
System：Serum-free DMEM：4ml；PLent-HIV-1-CD4-CAR plasmids：10μg；GM easyTM Lentiviral Mix：10μl
(10μg)；HG TransgeneTM Reagent：60μl.After mixing, after room temperature places 20min, drop evenly containing 293T
In Tissue Culture Dish, CO is placed on2Cultivated in incubator.After transfecting 24h, carefully sop up cell culture fluid and abandon in filling thimerosal
Waste liquid cup in, then plus 15ml contains the fresh culture medium of 10% serum and continues to cultivate.After changing liquid 48h, cell conditioned medium is drawn
Liquid is in 50ml centrifuge tubes, and 4 DEG C, 500g centrifugation 5min, supernatant is transferred in new centrifuge tube after being filtered with 0.45 μm of filter.This
When supernatant in virion can directly go detection titre.
By above-mentioned virus using TCID50 measure titres, by the 293T cells in exponential phase with 1 × 104Cells/
The amount in hole is inoculated in 96 porocyte culture plates, and sample presses 10 times of double dilution series concentration with 5%FBS DMEM, is loaded onto 96 holes
In cell, each concentration is loaded 10 holes, if 2 hole blank controls.Cultivated in 37 DEG C, 5%CO2, observe cell day by day and malicious spot occur
Situation, generally requires observation 5-7 days, and the TCID50 results of sample are calculated according to concentration and hole count that malicious spot occur.The result shows that weight
The titre 6.5 × 10 of group slow virus6TCID50/ml。
Embodiment 8
The preparation embodiment of PBMC cells.
The fresh peripheral blood of 75ml health contributors is taken, (is given birth to TBD sample rates separating liquid purchased from Tianjin Hao oceans China Tech
Thing), separating peripheral blood mononuclear cells PBMC methods are as follows：
(1) peripheral blood 75ml and physiological saline are pressed 1:1 dilution proportion.Blood after dilution is carefully added on an equal basis
On volume lymphocyte separation medium, obvious layering, room temperature horizontal centrifugal 800rpm/min, 20min are formed.In centrifuge tube certainly at this time
Upper and lower 4 layers of formation；Blood plasma, the tunica albuginea layer being made of PBMC, lymphocyte separation medium layer and nethermost erythroprecipitin layer.
(2) tunica albuginea layer is carefully drawn with suction pipe, all suctions out PBMC as far as possible.Add 2 times of amount physiological saline, wash cell 2 times,
Centrifugation, 800rpm/min, 10min after mixing every time.Low-speed centrifugal is conducive to remove the blood platelet retained in cell suspension and leaching
Bar cell separating liquid, supernatant discarding after centrifugation, collects PBMC cells.
Embodiment 9
The preparation embodiment of the BD magnetic bead sortings of CD8+T lymphocytes, culture and HIV-1-CD4-CAR-T.
1) isolated PBMC is washed 2 times with PBS.2) 300 × g of centrifugation cell, 10 minutes.3) cell is resuspended,
The moon for adding biotin coupling selects antibody (being purchased from BD companies).4) at room temperature, it is incubated 20 minutes.Then washed with PBS buffer
Cell, centrifugation cell 300 × g, 10 minutes.5) Avidin coupled bead (being purchased from BD companies) is added.6) at room temperature, it is incubated
30 minutes.7) cell is resuspended with PBS buffer, adds in streaming pipe, often pipe is no more than 3ml.8) streaming pipe is placed on cell point
Select on magnet stand, quiet 8 minutes, allow the non-CD8+T cells for being combined with magnetic bead to be adsorbed in side wall.9) collect not by enrichment with magnetic bead
Supernatant, centrifuges 300 × g, 10 minutes, what is obtained was CD8+T cells.
CD8+T cells are resuspended with RPMI1640 complete mediums, then by 1 × 106The cell concentration of/ml, uniformly spreads extremely
In Tissue Culture Plate.Then (it is purchased from using anti-CD3 (being purchased from BD companies), anti-CD28 (being purchased from BD companies) antibody and IL2
BD companies) cell is stimulated, effect 48 it is small when after, collect cell.Recombinant slow virus is infected in the ratio of MOI=10
CD8+T, fresh culture is changed after infecting 24h, continues to expand culture to enough dosages.(it is purchased from by FC500 flow cytometers
BECKMAN companies) FL1 Air conduct measurements Chimeric antigen receptor expression (Fig. 4).Using the CD8+T cells do not transformed as negative control,
HIV-1-CD4-CAR-T positive rates 81.8%.
Embodiment 10
HIV-1-CD4-CAR-T cells detect the neurological susceptibility of HIV-1
Wild type HIV-1NL4-3Pair unloaded to CD4+T lymphocytes, the CD8+T lymphocytes do not transformed, transduction respectively
Infected according to CD8+T cells, CD4- ζ CAR-T and HIV-1-CD4-CAR-T cells, wherein CD4+T lymphocytes are as experiment
Positive control.Then pass through the culture of 10 days, Thermo Fisher (are purchased from by HIV-1p24 antigen ELISA detecting kits
Scientific companies) analysis 5 experimental groups intracellular HIV-1 antigen p24 contents.The specific steps of ELISA detections：
Plus conjugate 1 A.：1,25 μ L of conjugate are added per hole.B. it is loaded：Sequentially add sample, negative control, p24 antigens
75 μ L of positive control.C. incubate：Microwell plate is sealed with sealing plate film, 37 DEG C is put and incubates 60 minutes.D. board-washing：It is anti-to discard microwell plate
Liquid in hole is answered, 350 μ L working concentration cleaning solutions are added per hole (containing blank control wells), then discard cleaning solution.Repetition is washed
Plate totally 5 times, finally pats dry.E. it is enzyme：Add conjugate 2 (blank control wells are not added with) per hole, microwell plate is sealed with sealing plate film, is put
37 DEG C incubate 30 minutes.F. board-washing：Liquid in microwell plate reacting hole is discarded, 350 μ L work is added per hole (containing blank control wells)
Concentration cleaning solution, then discards cleaning solution.Repeat board-washing totally 5 times, finally pat dry.Plus substrate solution G.：Add substrate buffer per hole
Liquid and each 50 μ L of color developing agent (containing blank control wells), are sealed microwell plate with sealing plate film, put room temperature (18~30 DEG C) Incubation in dark
30 minutes.Plus terminate liquid H.：Add 50 μ L of terminate liquid (containing blank control wells) per hole, gently vibrate microwell plate, make content abundant
Mix.I. detect：It is interior when 1 is small after termination reaction, with microplate reader at 450nm wavelength, each hole absorbance is measured, according to critical
Whether contain HIV-1p24 antigens in value judgement sample.
The CD4+T lymphocytes that the results show has more than half are positive for p24, and the CD4- ζ CAR-T cells for having 10% are
P24 is positive, and p24 positive cells ratio in HIV-1-CD4-CAR-T cells, with CD8+T lymphocytes, the transduction sky do not transformed
The control CD8+T cells of load are suitable, are p24 feminine genders.Therefore, HIV-1-CD4-CAR-T cells can invading to avoid HIV-1
Dye.
Embodiment 11
The HIV-1-CD4-CAR-T cells stimulated by HIV-1 envelope proteins can be with efficient secretion antiviral cell factor
By HIV-1-CD4-CAR-T, CD4- ζ CAR-T and CD8+T is not transformed respectively by E：T (effector cell and target cell
Than) it is 1：1 with expressing HIV-1NL4-3Envelope glycoprotein cell line Jurkat is mixed, and is tested and (be purchased from ELIspot
EBioscience companies) HIV-1-CD4-CAR-T, CD4- ζ CAR-T and point for not transforming CD8+T cell IFN-γ are detected respectively
Secrete.
ELIspot specific steps：
1st day：1. pre-coated plate of cell culture (sterile working) activates：RPMI-1640 culture mediums are added per hole, room temperature is quiet
Clappers remove culture medium after putting 5-10 minutes.2. add cell suspension：Cell suspension (effector cell and the target of concentration will be adjusted
The mixed cell suspension of cell) add each experimental port, 100 μ L/ holes；Positive control wells：Cell concentration 1 × 105/ hole, adds thin
Born of the same parents' culture medium and PHA；Negative control hole：Cell concentration 1 × 105/ hole, adds isometric cell culture medium；Background negative control：Add
Enter the RPMI-1640 culture mediums containing hyclone.3. it is incubated：37 DEG C are put into, 5%CO2When incubator culture 20 is small.
2nd day：(no longer needing sterile working) 4. cell lysis is operated after culture：Pouring aperture inner cell and culture medium.Add
The deionized water that ice bath is crossed, 200 μ L/ holes, 4 DEG C of refrigerators place 10 minutes hypotonic lysis cells.5. board-washing：Liquid in pouring aperture, 1
× Washing buffer, 200 μ L/ holes, are washed 5-7 times.30-60s is stood every time.For the last time, have the final say and go on blotting paper
Net liquid body.6. detect antibody incubation：The antibody-solutions of biotin labeling are added into plate hole, 100 μ L/ holes.When 37 DEG C of incubations 1 are small.
7. board-washing：Liquid in pouring aperture, 1 × Washing buffer, 200 μ L/ holes, are washed 5 times.Stand 30-60 seconds every time.Last
Secondary, clappers remove liquid on blotting paper.8. enzyme-linked Avidin is incubated：The enzyme mark avidin solution diluted is added into plate hole,
100 μ L/ holes.When 37 DEG C of incubations 1 are small.9. board-washing：Liquid in pouring aperture, 1 × Washing buffer, 200 μ L/ holes, washing 5
It is secondary.Stand 30-60 seconds every time.For the last time, clappers remove liquid on blotting paper.10. colour developing：The AEC nitrite ions that will now match somebody with somebody
Add each plate hole, 100 μ L/ holes.Room temperature lucifuge stands colour developing 25 minutes, 11. color development stoppings：Liquid in pouring aperture, opens plate bottom
Seat, is washed with deionized 3-5 times, color development stopping.Plate is placed on room temperature shady place, closes base after its naturally dry.
12.ELISPOT plate spots are taken pictures and are analyzed.
The result shows that：With expressing HIV-1NL4-3The HIV-1-CD4-CAR- of envelope glycoprotein cell line Jurkat mixed culture
The secretion capacity of T (left side) cell IFN-γ be significantly higher than CD4- ζ CAR-T (in) and do not transform CD8+T (right side) cell (Fig. 5).
Embodiment 12
The killing activity analysis of HIV-1-CD4-CAR-T cells
Express HIV-1NL4-3For envelope glycoprotein cell line Jurkat as target cell, effector cell is respectively HIV-1-CD4-
CAR-T, CD4- ζ CAR-T and CD8+T cells are not transformed.
By E：T is 1：1, add 1 × 106A Jurkat cell, after cell is completely adherent, collects HIV-1-CD4-CAR-
T, CD4- ζ CAR-T and CD8+T cells are not transformed, it is 1 × 10 to adjust cell concentration respectively7/ ml, adds 100 μ L per hole, 37 DEG C,
5% CO2Under the conditions of cultivate 12h.Abandon supernatant and add the 20 diluted CCK8 of μ L (being purchased from MCE companies), when incubation 4~6 is small, enzyme mark
Instrument detects the light absorption value of OD450.Killing rate=[1- (the OD values of effector cell+Target cell wells OD values-individual effect cell)/mono-
The OD values of only target cell] × 100%.HIV-1-CD4-CAR-T is bright for 90.56 ± 8.23% to the killing-efficiency of Jurkat cell
It is aobvious to be higher than CD4- ζ CAR-T and do not transform CD8+T cells (Fig. 6).
Embodiment 13
The analysis that AP1903 controls HIV-1-CD4-CAR-T cytoactives
1 × 10 is added per hole6HIV-1-CD4-CAR-T cells add 1 μ g AP1903 spies after cell is completely adherent
Heterogenetic antibody (is purchased from Apexbio companies), 37 DEG C, 5% CO2Under the conditions of cultivate 12h.Abandon supernatant and add the 10 diluted CCK8 of μ L
(being purchased from MCE companies), when incubation 4~6 is small, microplate reader detects the light absorption value of OD450.AP1903 is thin to HIV-1-CD4-CAR-T
Cytoactive control rate=[1- (add AP1903 specific antibodies hole OD values/AP1903 specific antibodies hole OD values are not added)]/
Positive rate=98.45 ± 6.27% of HIV-1-CD4-CAR-T.
From the above results, HIV-1-CD4-CAR molecules of the present invention, design is reasonable, safely and effectively, to treat HIV-1
Lay the foundation.
Embodiment 14
HIV-1-VRC01-CAR-T、HIV-1-VRC01b-CAR-T、HIV-1-VRC01-C-CAR-T、HIV-1-
The killing activity analysis of 3BNC60-CAR-T and HIV-1-NIH45-46-CAR-T cells
Express HIV-1NL4-3For envelope glycoprotein cell line Jurkat as target cell, effector cell is respectively HIV-1-
VRC01-CAR-T、HIV-1-VRC01b-CAR-T、HIV-1-VRC01-C-CAR-T、HIV-1-3BNC60-CAR-T、HIV-1-
NIH45-46-CAR-T, CD4- ζ CAR-T and CD8+T cells are not transformed.
By E：T is 1：1, add 1 × 106A Jurkat cell, after cell is completely adherent, collects HIV-1-VRC01-
CAR-T、HIV-1-VRC01b-CAR-T、HIV-1-VRC01-C-CAR-T、HIV-1-3BNC60-CAR-T、HIV-1-NIH45-
46-CAR-T, CD4- ζ CAR-T and CD8+T cells are not transformed, it is 1 × 10 to adjust cell concentration respectively7/ ml, 100 μ are added per hole
L, 37 DEG C, 5% CO2Under the conditions of cultivate 12h.Abandon supernatant and add the 20 diluted CCK8 of μ L (being purchased from MCE companies), it is small to be incubated 4~6
When, microplate reader detects the light absorption value of OD450.The killing rate=[1- (OD of effector cell+Target cell wells OD values-individual effect cell
Value)/individually target cell OD values] × 100%.HIV-1-CAR-T to the killing-efficiency of Jurkat cell up to more than 85%, it is bright
It is aobvious to be higher than CD4- ζ CAR-T and do not transform CD8+T cells (Fig. 7).
Embodiment 15
Prepare the kit of HIV-1-CD4-CAR-T cells
(1) carrier as described above for stablizing expression HIV-1-CD4-CAR is obtained；
(2) carrier diluent；
(3) operation instructions；
Embodiment 16
Application of the kit in treatment HIV-1
(1) carrier for stablizing expression HIV-1-CD4-CAR in kit is subjected to slow virus packaging：Using Lentiviral
Packaging Kit slow virus package kits, slow virus package cell line 293T is inoculated in containing DMEM+10%FBS
In 10cm culture dishes, 37 DEG C, cultivate under the conditions of 5% CO2, prepare transfection when adherent rate is 70%-80%.Take sterile
1.5ml EP are managed or 15ml centrifuge tubes, and reaction system is prepared by following component：Serum-free DMEM：4ml；pLent-HIV-1-CD4-
CAR plasmids：10μg；GM easyTM Lentiviral Mix：10μl(10μg)；HG TransgeneTM Reagent：60μl.
After mixing, after room temperature places 20min, drop evenly containing in 293T Tissue Culture Dish, be placed on CO2Cultivated in incubator.
After transfection 24, carefully sop up cell culture fluid and abandon in filling in the waste liquid cup of thimerosal, then add 15ml to contain 10% serum new
Fresh culture medium continues to cultivate.After changing liquid 48h, cell supernatant is drawn in 50ml centrifuge tubes, 4 DEG C, 500g centrifuges 5min, supernatant
Liquid is transferred in new centrifuge tube after being filtered with 0.45 μm of filter.
(2) fresh peripheral blood of 75ml health contributors is taken, (Tianjin Hao oceans China Tech is purchased from TBD sample rates separating liquid
Biology), separating peripheral blood mononuclear cells PBMC, method is as follows：
Peripheral blood 75ml and physiological saline are pressed 1:1 dilution proportion.Blood after dilution is carefully added in peer
On product lymphocyte separation medium, obvious layering, room temperature horizontal centrifugal 800rpm/min, 20min are formed.At this time from upper in centrifuge tube
And lower 4 layers of formation；Blood plasma, the tunica albuginea layer being made of PBMC, lymphocyte separation medium layer and nethermost erythroprecipitin layer.
Tunica albuginea layer is carefully drawn with suction pipe, all suctions out PBMC as far as possible.Add 2 times of amount physiological saline, wash cell 2 times, often
Centrifugation, 800rpm/min, 10min after secondary mixing.Low-speed centrifugal is conducive to remove the blood platelet and lymph retained in cell suspension
Cell separating liquid, supernatant discarding after centrifugation, collects PBMC cells.
(3) the BD magnetic bead sortings of CD8+T lymphocytes, culture and the preparation 1 of HIV-1-CD4-CAR-T) will be isolated
PBMC washed 2 times with PBS.2) 300 × g of centrifugation cell, 10 minutes.3) cell is resuspended, adds the cloudy choosing of biotin coupling
Antibody (is purchased from BD companies).4) at room temperature, it is incubated 20 minutes.Then cell, centrifugation cell 300 are washed with PBS buffer
× g, 10 minutes.5) Avidin coupled bead (being purchased from BD companies) is added.6) at room temperature, it is incubated 30 minutes.7) PBS buffer is used
Cell is resuspended, adds in streaming pipe, often pipe is no more than 3ml.8) streaming pipe is placed on cell sorting magnet stand, quiet 8 minutes, allows knot
The non-CD8+T cells that closing has magnetic bead are adsorbed in side wall.9) collect not by the supernatant of enrichment with magnetic bead, centrifuge 300 × g, 10
Minute, what is obtained is CD8+T cells.
CD8+T cells are resuspended with RPMI1640 complete mediums, then by 1 × 106The cell concentration of/ml, uniformly spreads extremely
In Tissue Culture Plate.Then (it is purchased from using anti-CD3 (being purchased from BD companies), anti-CD28 (being purchased from BD companies) antibody and IL2
BD companies) cell is stimulated, effect 48 it is small when after, collect cell.Recombinant slow virus is infected in the ratio of MOI=10
CD8+T, fresh culture is changed after infecting 24h, continues to expand culture to enough dosages.
It should be appreciated that the purposes of these embodiments is merely to illustrate the present invention and is not intended to the protection model of the limitation present invention
Enclose.In addition, it should also be understood that, after the technology contents of the present invention have been read, those skilled in the art can make the present invention each
Kind change, modification and/or variation, all these equivalent forms equally fall within the guarantor that the application the appended claims are limited
Within the scope of shield.
Claims (10)
1. for the Chimeric antigen receptor gene of HIV-1, it is characterised in that：The gene includes guiding sub-district, the CD4 with gp120
Domain, hinge area, transmembrane domain, costimulation domain, signal transduction domain and the suicide gene that binding site combines
System area.
2. the Chimeric antigen receptor gene of HIV-1 is directed to as claimed in claim 1, it is characterised in that：It is described and gp120
The domain that CD4 binding sites combine is using appointing in T4 antigen, VRC01, VRC01b, VRC01-C, 3BNC60, NIH45-46
One.
3. the Chimeric antigen receptor gene of HIV-1 is directed to as claimed in claim 2, it is characterised in that：It is described and gp120
The domain that CD4 binding sites combine uses T4 antigen.
4. the Chimeric antigen receptor gene of HIV-1 is directed to as claimed in claim 3, it is characterised in that：It is described guiding sub-district be
CD8 guides sub-district；The hinge area of the CAR is CD8 hinge areas；The transmembrane domain of the CAR is CD8 transmembrane regions；The CAR
Costimulation domain be 4-1BB functional domain；The signal transduction domain of the CAR includes the functional structure of CD3 ζ
Domain；The suicide gene system of the CAR is iCasp9 suicide gene systems.
5. the Chimeric antigen receptor gene of HIV-1 is directed to as claimed in claim 1, it is characterised in that：It is described to be directed to HIV-1's
The nucleotide sequence of Chimeric antigen receptor gene such as SEQ ID NO.1.
A kind of 6. plasmid, it is characterised in that：The plasmid is included if claim 1-5 any one of them is for the embedding of HIV-1
Close antigen receptor gene.
7.T cells, it is characterised in that：The T cell is included if claim 1-5 any one of them is for the chimeric of HIV-1
Antigen receptor gene.
8. a kind of kit, including
Stablize the carrier that expression is directed to the Chimeric antigen receptor gene of HIV-1 as described in claim any one of 1-5；
And carrier diluent.
9. as Chimeric antigen receptor gene of the claim 1-5 any one of them for HIV-1 is preparing the medicine for the treatment of HIV-1
Application in thing.
10. application as claimed in claim 9, it is characterised in that：The form of the medicine includes kit.
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