CN106480022A - Interfere fragment and its application - Google Patents
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Abstract
The invention discloses interfere fragment and its application, the nucleotide sequence that the action target spot of the wherein interference fragment is as follows：5′-GCCTGTGTTCTCTGTGGACTA-3′(SEQ ID NO：5).The interference fragment is good for above-mentioned target spot targeting, interference inhibition to PD-1 is projected, and, above-mentioned interference fragment is proceeded to immunocyte, the expression of immunocyte PD-1 can be made to be suppressed for a long time, obtain the immunocyte of PD-1 expression inhibiting, and the expression of the proliferative cell PD1 that the cell is obtained after entering human body is also suppressed, and then the immunocyte of the PD-1 expression inhibiting for obtaining can be effective for preparing immunotherapy medicaments, and then it is used for the immunization therapy of tumour, so as to immunotherapeutic effects are effectively improved, reduce side effect.
Description
Technical field
The present invention relates to immuno-biology technical field, and in particular to interfere fragment and its application.
Background technology
Malignant tumour is the main fatal disease type of the mankind, and the death rate remains high.Ministry of Public Health's recent statistics as shown by data is disliked
Property tumour to occupy China resident top ten cause of the death the first.Chinese 2,680,000 people of Incidence patient in 2010, dead 1,970,000
People；The whole nation has more than 600 ten thousand people of malignant tumor patient at present.It is total that the annual medical expense for being used for malignant tumor patient accounts for health
The 20% of expense, is the maximum medical burden of the whole society.Current treatment means for reach healing, extend malignant tumor patient
The effect of target such as life cycle and quality of making the life better is still extremely limited, even particularly with certain form of malignant tumour
Early diagnosis is also difficult to obtain satisfied curative effect, and the malignant tumour as metastatic, general or progressive stage is then more difficult to cure.
In current ideas of cancer therapy, the local treatment such as operation and radiotherapy has preferable curative effect to limitation tumour, and for complete
MRD after body, metastatic or local treatment then relies primarily on chemotherapy or immunotherapy and carries out systemic treatment.Swollen
Knurl morbidity is multi-step, the result of polygenes effect, and tumour cell shows to grow, it is out of control with apoptosis to break up.Clinically sick
The diversity of people's symptom and individual inheritance are heterogeneous, and the appearance of tumour DISTANT METASTASES IN focus, make the oncotherapy must be with whole body
The viewpoint of property disease, using the comprehensive therapeutic plan of individuation.I.e. according to the feature of each tumour patient, tied using local treatment
Close whole body therapeutic；The tumour of local lesion will not only be eliminated, to also control tumour Preventive grow and tumour to important dirty
The invasion and attack of device, could really effectively improve cure rate and the life cycle of tumour patient.The treatment of generally tumor by local focus can be adopted
Take operation and physical treatment (involving laser therapy etc. including RF ablation, thermotherapy, radiotherapy, freezing, ultrasound) method eliminated,
But for the not detectable tumour minimal disease of iconography (in the residual after transfer and relapse stove, local treatment, blood
Cancer cell), above local treatment means are then helpless, it is necessary to solved by systemic treatment means.Due to tumour
Transfer, recurrence, spread more fatal, therefore systemic treatment be particularly important with urgent.Systemic treatment has biography at present
System chemotherapy, molecular targeted agents treatment, biological therapy (including cellular replacement therapy and immune cell therapy) and base
Because for the treatment of etc..Although classic chemotherapy is had made some progress in terms of some oncotherapies, to extending tumour patient life cycle side
Face is still contributed less.And natural drug resistance and Acquired lung infection and chemotherapeutic toxicity of the tumour cell to chemotherapeutic, making
The development for learning treatment receives serious obstruction.Clinical practice for many years proves that it is not the strong event of whole body therapeutic.
Immune cell therapy is that recent two decades grow up, as its therapeutic domain is wide (can be used for entity tumor and leukaemia),
Especially tumour minimal disease (including the cancer cell in transfer, recurrence stove, blood) more effectively, is had no toxic side effect, and
Suitable for tumour each stage (as late period Radiotherapy chemotherapy can not be used), therefore it is a strong event of whole body therapeutic.Immunity
Cell therapy has significant effect to improving quality of life, prolongation life cycle, extremely praises highly both at home and abroad, is current whole body therapeutic
One of best means, clinical potentials are huge.
The programmed death factor 1 (programmed cell death-1, PD-1) is belonging to 28 (costimulatory of costimulatory molecules
Molecule28, CD28)/cytotoxic t lymphocyte-associated antigen 4 (cytotoxic lymphocyte associated antigen,
CTLA-4) the Inhibitory receptor of family.The mark of PD-1 not still programmed death cell, at the same be expressed in bone-marrow-derived lymphocyte,
T lymphocyte, NK cell, NKT cell and Macrophage Surface.Recent study is even more and finds, by antigenic activation
T lymphocytic cell surface can briefly express PD-1, and the continuous expression of PD-1 may result in T cell dysfunction.PD-1
The popularity of expression and immune suppression function so as to become the focus of immunization therapy in recent years.In cancer, autoimmunity disease, slow
In the treatment of the diseases such as infections, PD-1 all plays an important role.At present, the application of main PD-1 is mainly showed
For the antibody of exploitation specificity closing PD-1 signal, then such antibody drug is used for controlling for TCA and metastatic cancer
Treat, although therapeutic effect still, but also often has the related adverse events of medicine and occurs.
Thus, application of the current PD-1 in terms of immunization therapy still has much room for improvement.
Content of the invention
Firstly, it is necessary to illustrate, the present invention is completed based on the following discovery of inventor：
The antibody drug of specificity closing PD-1 signal is used for the treatment of TCA and metastatic cancer, although therapeutic effect
Still, but the total incidence of the related adverse events (adverse events, AEs) of medicine is 41%, 3/4 grade of severe drug AEs
For 5%, mainly include：Skin (16%), gastrointestinal reaction (12%), lung's (7%).The related pneumonia incidence of medicine
For (6%), 3 grades/4 grades pneumonia incidences are 2% (2/129), and test early stage has 2 patients dead because of pneumonia.
Also, some patients have response for Antybody therapy, also have some patients after Antybody therapy is received its indices nothing bright
Aobvious change.Some patients response after Antybody therapy is received is incomplete, the such as male patient of a multiple organ canceration in 72 years old,
After the treatment of a course for the treatment of, the canceration metastatic of pancreas substantially reduces, but other site cancerations are nothing significant change.Have
Patient for Antybody therapy response completely, there is tumour regression.Additionally, some cases are multiple for this Antybody therapy response situation
Miscellaneous, the female patient of the such as many places scrofula liver canceration of 52 years old, after Antybody therapy is received, many places canceration
Decline still has lymph node canceration to strengthen.
The antibody of closing PD-1 signal path is not to directly act on tumor tissues but is made by activating antineoplastic immune and playing
With.Due to complexity and the individual difference of immune response, the therapeutic effect of these antibody in different types of cancer patient and
Differ greatly between Different Individual.As it was previously stated, the side effect that PD-1 inhibitor (antibody) is produced after venous re-transfusion is
Exist, the patient of simultaneity factor immunological diseases should not use the medicine, because PD-1 inhibitor can strengthen killing for immunocyte
Hinder ability, increase immune response, aggravation.Also after having some patients' Antybody therapy, response is incomplete.
It is contemplated that at least solving one of technical problem present in prior art.For this purpose, it is an object of the present invention to carrying
Go out a kind of means that can effectively suppress immunocyte PD-1 to express, thus, by suppressing the expression of PD-1 in immunocyte,
And then be used for preparing immunotherapy medicaments by the immunocyte of the PD-1 expression inhibiting of acquisition, and the medicine is used for exempting from for tumour
Epidemic disease is treated such that it is able to effectively solving the problems referred to above, improves range of application and application effect of the PD-1 in immunization therapy.
Specifically, in order to solve the above problems, the present invention utilizes technique for gene engineering, successfully constructs the shRNA for PD-1
Slow virus carrier, and screen the target sequence for obtaining jamming effectiveness more than 90%；Then, the interference target for being obtained based on screening
Point sequence, by carrying the corresponding pd-1shRNA slow virus carrier for interfering fragment, the corresponding interference fragment of screening is proceeded to and is exempted from
Epidemic disease cell T lymphocyte (includes：CTL, TIL, CD8+T, NKT cell etc.) so as to the expression of PD-1 is subject to
Long-term suppression, obtains the immunocyte of PD-1 expression inhibiting, the proliferative cell PD1's that wherein cell is obtained after entering human body
Expression is also suppressed；Further, such T cell through modification is fed back human body or tumor by local by inventor, is as a result found, energy
Immunotherapeutic effects are enough effectively improved, reduces side effect to the full extent.
Thus, according to an aspect of the present invention, the invention provides a kind of interfere fragment.Embodiments in accordance with the present invention, should
The nucleotide sequence for interfering the action target spot of fragment to be as follows：
5′-GCCTGTGTTCTCTGTGGACTA-3′(SEQ ID NO：5).
It is surprisingly found by the inventors that, the interference fragment of the present invention is good for above-mentioned target spot targeting, the interference suppression effect to PD-1
Fruit is prominent.Also, embodiments in accordance with the present invention, above-mentioned interference fragment is proceeded to immunocyte T lymphocyte (for example
CTL, TIL, CD8+T, NKT cell etc.), the expression of immunocyte PD-1 can be made to be suppressed for a long time, obtained
The immunocyte of PD-1 expression inhibiting, and after the cell enters human body, the expression of the proliferative cell PD1 of acquisition is also suppressed,
So as to the immunocyte of the PD-1 expression inhibiting of the acquisition effective for preparing immunotherapy medicaments, and then can be used for tumour
Immunization therapy, effectively improve immunotherapeutic effects, reduce side effect to the full extent.
In addition, according to the above embodiment of the present invention interfere fragment have following additional technical characteristic：
Embodiments in accordance with the present invention, the interference fragment are made up of the positive-sense strand with following nucleotide sequence and antisense strand：
Positive-sense strand：5′-GATCCGCCTGTGTTCTCTGTGGACTATTCAAGAGATAGTCCACAGAGAA
CACAGGCTTTTTTG-3′(SEQ ID NO：6),
Antisense strand：5′-AATTCAAAAAAGCCTGTGTTCTCTGTGGACTATCTCTTGAATAGTCCAC
AGAGAACACAGGCG-3′(SEQ ID NO：7).
According to a further aspect in the invention, present invention also offers a kind of method that PD-1 is expressed in suppression immunocyte.According to
Embodiments of the invention, the method include：Foregoing interference fragment is proceeded to the immunocyte.
Inventor is had found, can effectively be suppressed the expression of PD-1 in immunocyte using the method, obtain PD-1 expression inhibiting
Immunocyte.Also, obtain PD-1 expression inhibiting immunocyte can effective for the preparation of immunotherapy medicaments,
And then it is obtained in that therapeutic effect is good, the low immunotherapy medicaments of side effect；Additionally, the immunocyte of the PD-1 expression inhibiting
Can also be used directly to feed back human body or tumor by local such that it is able to effectively treatment or prevention of tumor.
Embodiments in accordance with the present invention, in the method for the invention, the method for interfering fragment to proceed to immunocyte are not especially limited
System.According to some specific examples of the present invention, the interference fragment is proceeded to the immunocyte using slow virus carrier system.
Thus, interfere fragment to be easy to proceed to immunocyte such that it is able to interference efficiency is effectively improved, realize to immunocyte PD-1 table
The suppression for reaching.
Embodiments in accordance with the present invention, the immunocyte are lymphocyte, preferably T cell, B cell, NK cell, NKT
Cell, more preferably tumor-infiltrated T lymphocyte or CTL cell.Thereby, it is possible to effectively obtain the immunity of PD-1 expression inhibiting
Cell, and then, the immunocyte of the PD-1 expression inhibiting for obtaining is used for the preparation of immunotherapy medicaments, effectively can be obtained
Therapeutic effect is good, the low immunotherapy medicaments of side effect.Can be directly used for；The immunocyte of the PD-1 expression inhibiting also may be used
To be used directly to feed back human body or tumor by local such that it is able to effectively treatment or prevention of tumor.
, wherein it is desired to illustrate, the term " immunocyte " for herein being adopted is primarily referred to as the lymph in blood of human body
Cell, such as T lymphocyte, bone-marrow-derived lymphocyte, NK lymphocyte, NKT lymphocyte.Wherein, T lymphocyte can
Think tumor-infiltrated T lymphocyte, and through in vitro culture or the various T cell of induction, such as CTL cell.Term
" CTL cell " in this article refers to mix by DC cell loading tumour antigen and with autologous lymphocyte, stimulates autologous pouring
The cytotoxic T lymphocyte that bar cell is produced.
According to another aspect of the invention, present invention also offers foregoing interference fragment is in immunotherapy medicaments are prepared
Purposes.Embodiments in accordance with the present invention, the immunocyte comprising PD-1 expression inhibiting in the medicine.Thus, tumour is suffered from
Person gives the medicine, can effectively suppress the expression of PD-1 in patient tumors cell, so as to effectively treatment tumour；In addition the medicine
Thing can also be effective for the prevention of tumour, and the effect for the treatment of or prevention of tumor is good, and side effect is low.
Embodiments in accordance with the present invention, the immunocyte of the PD-1 expression inhibiting is through the following steps that obtain：Will above
Described interference fragment proceeds to the immunocyte, to obtain the immunocyte of PD-1 expression inhibiting.Thereby, it is possible to efficient
Ground obtains the immunocyte of PD-1 expression inhibiting.
The interference fragment is proceeded to the immunocyte using slow virus carrier system by embodiments in accordance with the present invention.Thus,
Fragment is interfered to be easy to proceed to immunocyte, interference efficiency is high, the inhibition to immunocyte PD-1 expression is good.
Embodiments in accordance with the present invention, the immunocyte are lymphocyte, preferably T cell, B cell, NK cell, NKT
Cell, more preferably tumor-infiltrated T lymphocyte or CTL cell.Thereby, it is possible to effectively obtain the immunity of PD-1 expression inhibiting
Cell, and then, the immunocyte of the PD-1 expression inhibiting can be used in the preparation of immunotherapy medicaments.
According to a further aspect in the invention, present invention also offers a kind of immunotherapy medicaments.Embodiments in accordance with the present invention, should
Immunocyte of the immunotherapy medicaments comprising PD-1 expression inhibiting, the immunocyte of the PD-1 expression inhibiting be by above
In the method suppression immunocyte of PD-1 expression in described suppression immunocyte, PD-1 expression is obtained.Thus, to tumour
Patient gives the immunotherapy medicaments of the present invention, can effectively suppress the expression of PD-1 in patient tumors cell, so as to effectively control
Treat tumour；In addition the medicine can also be effective for the prevention of tumour, and the effect for the treatment of or prevention of tumor is good, and side effect is low.
The additional aspect and advantage of the present invention will be set forth in part in the description, and partly will become bright from the following description
Aobvious, or recognized by the practice of the present invention.
Description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will be apparent from from the description with reference to accompanying drawings below to embodiment and
Easy to understand, wherein：
Fig. 1 is shown according to one embodiment of the invention, the structural representation of LV3 carrier；
Fig. 2 shown according to one embodiment of the invention, the PD-1 albumen of the aim cell of each shRNA slow virus carrier transfection
Western testing result；And
Fig. 3 shows that according to one embodiment of the invention PD-1 is positive in the aim cell of each shRNA slow virus carrier transfection
The flow cytomery result of cell quantity.
Specific embodiment
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that the following examples
The present invention is merely to illustrate, and be should not be taken as limiting the scope of the invention.In embodiment, unreceipted particular technique or condition, press
(for example write with reference to J. Pehanorm Brooker etc., Huang Peitang etc. is translated according to the technology described by document in the art or condition《Molecule
Cloning experimentation guide》, the third edition, Science Press) or carry out according to product description.Agents useful for same or instrument unreceipted
Production firm person, be can by city available from conventional products, can for example purchase from Illumina company.
Embodiment 1
First, plasmid construction：
Control NC (negative control) is set, in accordance with the following methods, builds the shRNA slow virus carrier of three kinds of PD-1：
(1), experiment material
1st, DNA oligo is designed using Designer3.0 (Genepharma) software, and synthetic primer is by Shanghai Ji agate system
Medicine Technology Co., Ltd. completes
2nd, the instrument list used in testing：
3rd, the reagent list used in testing：
(2), flow process is built
1、Oligo Designer3.0
During inventor's design template, the loop structure in LV3-shRNA template has selected TTCAAGAGA to avoid the formation of
Termination signal；5 ' ends of positive-sense strand template with the addition of GATCC, with BamHI digestion after the cohesive end that formed complementary；Antisense strand mould
5 ' ends of plate with the addition of AATTC, with EcoRI digestion after the cohesive end that formed complementary, specific as follows：
Positive-sense strand：
5’-GATCC-(GN18)-(TTCAAGAGA)-(N18C)-TTTTTTG-3’；
Antisense strand：
3’-G(CN18)-(AAGTTCTCT)-(N18G)-AAAAAACTTAA-5’.
Wherein, the specifying information of the shRNA slow virus carrier of control shNC and three kinds of PD-1 is as follows：
(1), the specifying information of control shNC is：
Target sequence：5’-TTCTCCGAACGTGTCACGT-3’(SEQ ID NO：1)；
Sense strand sequence：
GATCCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACT
TTTTTG(SEQ ID NO：2)；
Antisense strand sequence：
AATTCAAAAAAGTTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGG
AGAACG(SEQ ID NO：3)；
Transcription product sequence：
GTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACTT(SEQ
ID NO：4).
The shRNA slow virus carrier of (2) three kinds of PD-1：PDCD1-Homo-694, PDCD1-Homo-791 and
The plasmid information of PDCD1-Homo-238 is respectively as shown in table 1-3.
2nd, the annealing of LV3-shDNA template
DNA oligo is dissolved with TE (pH8.0) respectively, concentration is 100 μM.Take corresponding positive-sense strand and antisense strand oligo
Solution, configures annealing reaction system according to following proportioning：
| Component | Volume (μ l) |
| 10X shDNA annealing buffer | 5 |
| Positive-sense strand (100 μM) | 5 |
| Antisense strand (100 μM) | 5 |
| ddH2O | 35 |
| Cumulative volume | 50 |
Made annealing treatment according to following program in PCR instrument：95℃5min；85℃5min；75℃5min；70℃
5min；4 DEG C of preservations.The shRNA template that concentration is 10 μM is obtained after annealing.Gained template solution is diluted 50 times,
Final concentration of 200nM, for coupled reaction.
3rd, the linearisation of LV3 carrier
10 μ g LV3 carriers are taken, and digestion process are carried out according to following system：
| Component | Volume (μ l) |
| 2×Buffer Tango | 10 |
| BamHI | 5 |
| EcoRI | 5 |
| LV3 | 10μg |
| ddH2O | Mend to 100 |
| Cumulative volume | 100 |
37 DEG C of digestions 1 hour, agarose electrophoresis, is reclaimed using Agarose Gel DNA Purification Kit Ver2.0, electricity
Swimming detection estimated concentration, diluted concentration to 50ng/ μ l.
4th, the structure of LV3-shRNA carrier
The coupled reaction of carrier is carried out according to following system：
| Component | Volume (μ l) |
| 10 × T4 connects buffer solution | 2 |
| LV3(BamHI+EcoRI) | 1 |
| ShDNA template (100nM) | 1 |
| T4DNA ligase (5weissU/ μ l) | 1 |
| ddH2O | 15 |
| Cumulative volume | 20 |
22 DEG C 1 hour, convert to Escherichia coli Top10 (transform to Top10competent cells).
5th, the preparation of competent cell：(Calcium Chloride Method)
Competent cell is prepared using Calcium Chloride Method, comprise the following steps that：
5.1, from one single bacterium colony of picking in 37 DEG C of cultures fresh plate of 16 hours, go to one and cultivate containing 100ml LB
In the 1L flask of base.Culture 3 hour (rotary shaker, 300 rev/min) is acutely shaken in 37 DEG C.
Bacterium is aseptically transferred to an aseptic, disposable, ice-cold 50ml PA tube by 5.2
In, place 10 minutes on ice, make culture be cooled to 0 DEG C.
5.3 in 4 DEG C, are centrifuged 10 minutes with 4000 revs/min, reclaim cell.
5.4 pour out nutrient solution, pipe is inverted 1 minute, flows to end the trace nutrient solution of final residual.
5.5 with the ice-cold 0.1mol/L CaCl of 10ml2Resuspended per part of precipitation, is positioned on ice bath.
5.6 in 4 DEG C, are centrifuged 10 minutes with 4000 revs/min, reclaim cell.
5.7 pour out nutrient solution, pipe is inverted 1 minute, flows to end the trace nutrient solution of final residual.
5.8 per the 50ml initial incubation thing ice-cold 0.1mol/L CaCl of 2ml2(containing 20% glycerine) resuspended every part of cell
Precipitation.
Cell is distributed into aliquot (100 μ l/ are propped up) by 5.9, be put in -70 DEG C frozen.
6th, the conversion of connection product
Follow the steps below the conversion of connection product：
Competent cell is taken out in 6.1 from -70 DEG C, and the centrifuge tube that will be equipped with competent cell is placed 4 minutes on ice, treats competence
After cell thaws, 10 μ l connection products are added, softly content is mixed, place 30 minutes in ice.
Centrifuge tube is put on the rack for test tube that pre-heating is put well in 42 DEG C of water-bath by 6.2, place 90 seconds, should not shake from
Heart pipe.
6.3 are quickly transferred to centrifuge tube in ice bath, so that cell is cooled down 3 minutes.
6.4 to every centrifuge tube adds 800 μ l LB culture medium (without antibiotic), then centrifuge tube is transferred to 37 DEG C of shaking tables,
250 revs/min, cultivating makes bacteria resuscitation in 45 minutes.
6.5 take the cell after 200 μ l are cultivated is spread evenly across containing on 50 μ g/ml Ampicillin LB flat boards.
After liquid is absorbed on 6.6 grade flat boards, flat board is inverted in 37 DEG C of incubators, cultivates 16 hours.
7th, the identification of positive colony and sequencing
According to following steps evaluation and screening positive colony, and sequence verification is carried out to which：
7.1 5 bacterium colonies of picking, the LB cultures being inoculated into containing 50 μ g/ml Ampicillin (ampicillin) from every piece of flat board
In base, 37 DEG C are cultivated 16 hours.
7.2 extract plasmid using alkaline lysis.
7.3 gained plasmids carry out single endonuclease digestion identification with EcoRI.Digestion result shows that cut by EcoRI is probably positive gram
Grand, sequencing identification will be carried out per group clone.Sequencing result is shown in Table 1-3.
7.4 will be sequenced correct bacterial strain using the extracting of extraction agent box is measured in high-purity plasmid, and gained plasmid can be used for conventional
Molecular biology experiment and cytologic experiment.If cytotoxicity is larger when for cell transfecting, please convert again to large intestine bar
In bacterium Top10, then higher purity plasmid is prepared with kit or CsCl supercentrifugation.
Table 1, PDCD1-Homo-694 plasmid information
Table 2, PDCD1-Homo-791 plasmid information
The plasmid information of table 3, PDCD1-Homo-238
2nd, Sites Screening
(1) slow virus carrier prepares：
Follow the steps below slow virus carrier preparation：
1st, the slow virus incasing cells transfection 293T cell of Trypsin Induced exponential phase, is taped against in 6 orifice plates, 37 DEG C,
50mL/L CO2Culture in incubator, cell density are transfected when reaching 60%～70%. the front PBS cell of transfection 2 times.
2nd, 3 kinds of plasmid DNA solutions (VSVG 2 μ g, P8.7.4 μ g, 10 μ g of genes of interest) in slow virus packaging system are prepared.
3rd, sterilized water is settled to 25 μ L, adds CaCl2(2.5mol/L) 25 μ L of solution, mixes, and 4 degree are placed 20 on ice
min.
4th, 2 × HBS buffer salt solution 50 μ L, room temperature 30min are added dropwise over.
5th, DNA calcium phosphate mixed liquor is transferred in the nutrient solution containing cell monolayer, mix, culture 16h after discard containing
The nutrient solution of transfection mixture, is substituted with fresh nutrient solution.
6th, transfect after collect within 48 hours cell supernatant (1.5ml).
(2) prepared by aim cell：
1st, the separation of PMNC
The cell that obtain will be separated from the 100ml peripheral blood of the positive liver cancer patient collection of 1 AFP, be transferred to 50ml from
(1800rpm, 10min) is centrifuged in heart pipe, supernatant is sucked, is taken the liquid after supernatant and is slowly added in lymphocyte separation medium Ficoll
On liquid (GE), volume ratio is 1:1, it is centrifuged (2000rpm, 20min)；
Then, the white flock cell for collecting interface adds PBS, and gently piping and druming is mixed, and is centrifuged (1500rpm, 10min)；
Next, repeated centrifugation washs totally 3 times；
Then, collect cell and mixing is gently blown and beaten, add basal medium 20ml piping and druming uniform, be transferred to 75cm2Blake bottle
In, in 37 DEG C, 5%CO2Incubator culture 2h.
2nd, lymphocyte and monocytic separation
Take out 75cm2Blake bottle, gently by 75cm2Blake bottle straighten vertical, draw 18ml nutrient solution in new 175cm2In bottle,
Activation culture for use in lymphocyte；
For former bottle (75cm2Blake bottle) in remaining 2ml raffinate, will be used for for monocyte being induced to differentiate into DC cell.
3rd, the culture of lymphocyte
In the 175cm for cultivating lymphocyte2In bottle, 20ml CTL1 cell culture medium is added, after 24h, adds 20ml
CTL2 medium culture, then every 2～3d CTL3 culture medium equivalent fluid infusion.
Wherein, the compound method of CTL1 culture medium, CTL2 culture medium and CTL3 culture medium is：
CTL1 culture medium：The 1 pipe CTL1 factor is taken out from -20 degree, normal temperature melts, and is added in 20ml basal medium,
And add the serum of 10% client, wherein will 0.22um syringe needle filter filter after use.
CTL2 culture medium：The 1 pipe CTL2 factor is taken out from -20 degree, normal temperature melts, and is added in 20ml basal medium,
Wherein will use after the filtration of 0.22um syringe needle filter.
CTL3 culture medium：The 1 pipe CTL3 factor is taken out from -20 degree, normal temperature melts, and is added in 500ml basal medium,
Wherein will use after the filtration of 0.22um syringe needle filter, whole incubation is managed with 3.
The CTL1 factor (including IFN-γ, 1000U/ml)：1ml, -20 DEG C long-term preserve, and 4 DEG C preserve 2 weeks.
The CTL2 factor is (including IL-1,1000U/ml；IL-2,1000U/ml；CD3 antibody, 100ng/ml；CD28 antibody,
100ng/ml)：1ml, -20 DEG C long-term preserve, and 4 DEG C preserve 2 weeks.
The CTL3 factor is (including IL-2,1000U/ml；IL-7,20ng/ml；IL-15,20ng/ml)：1ml, -20 DEG C long
Phase preserves, and 4 DEG C preserve 2 weeks.
Thus, aim cell is obtained.Then, aim cell is divided in 6 orifice plates, per hole 106Cell, standby.
(3) viral vector infection：
1st, the vial supernatant of collection is added in the above-mentioned aim cell for preparing, is simultaneously introduced the fresh training of equal volume
Foster base (CTL3 culture medium) carries out culture aim cell (common 3ml).
2nd, liquid is changed after 24 hours, normal culture.
3rd, after infecting 96 hours, cell, leach protein are collected.
(4) PD-1 albumen western identification：
1st, reagent
1)0.01M PBS(pH7.4)：NaCl 8.0g；KCl 0.2g；Na2HPO41.44g；KH2PO40.24g；
Plus ddH2O to 1000ml.
2) protease inhibitors：
Protease Inhibitor Cocktail：Calbiochem company, article No. 539134
3)RIPA：
137mM NaCl,1.4mM KH2PO4,1.7mM KCl,
5.0mM EDTA,5.0mM EGTA,4.3mM Na2HPO4.
0.1%SDS, 1%TritonX-100,1mMNaVO4,50mM NaF.
4) Bio-Rad company Bradford dye liquor, article No. 500-0006.
5) 10%SDS-PAGE separation gel, concentrates glue,
Wherein, 10ml separation gel is prepared according to following formula：
ddH2O 4.0ml；30% acrylamide 3.3ml；1.5M Tris(pH8.8)2.5ml；
10%SDS 0.1ml；10% ammonium persulfate 0.1ml；TEMED 0.004ml.
Prepare according to following formula and concentrate glue 2ml：
ddH2O 1.4ml；30% acrylamide 0.33ml；1M Tris(pH6.8)0.25ml；
10%SDS 0.02ml；10% ammonium persulfate 0.02ml；TEMED 0.002ml.
6) 2X SDS-PAGE Loading Buffer：
7) albumen marker, Bio-Rad company, article No. 161-0324.
8) electrophoresis liquid：
Join 5X Tris-Glycine Buffer liquid storage, dilute during use.
Liquid storage：0.125M Tris, 1.25M Glycine, 0.5% (W/V) SDS,
Its compound method is as follows：
Weigh Tris 15.1g；Glycine 94g；SDS 5.0g, is placed in the beaker of 1L, be subsequently adding about 800ml go from
Sub- water, stirring and dissolving, after adding deionized water to be settled to 1L, room temperature preservation.
9) pvdf membrane, Millipore company, article No. IPVH00010.
10) transferring film buffer solution (transfer buffer)：
Glycine 2.9g；Tris 5.8g；SDS 0.37g；Methyl alcohol 200ml；Plus ddH2O is settled to 1000ml.
11) TBST compound method：
Claim NaCl 8g；KCl 0.2g；Tris 3g, is dissolved in 800ml distilled water, plus HCl adjusts pH to 7.4, constant volume to 1
Rise.Then plus 1ml Tween20, mix.
12) blockade liquid：TBST containing 5% skimmed milk power.
13) ECL reaction substrate, Pierce company, supersignal west pico chemilumiscent sunstrate, article No.
prod#34080.
2nd, instrument
1) protein concentration, Eppendorf company Biophotometer are determined；
2) electrophoresis and transferring film Bio-Rad powerpac 3000；
3rd, key step
1) preparation of samples
PBS cell.After removing PBS plus containing protease inhibitors (1:200) total protein extraction agent RIPA, splits on ice
Solution 30min.Cell is scraped with cell, be drawn in 1.5ml EP pipe, then 4 DEG C, 13,000g centrifugation 15min.Take
Supernatant is used as sample.Bradford colorimetric method for determining protein concentration.
2) electrophoresis：Running gel is prepared, carries out SDS-PAGE.
PAGE glue is prepared in advance.
Take the protein sample of phase homogenous quantities, plus isopyknic 2 × loading buffer, 5min in boiling water bath.
Loading, and albumen marker, unnecessary empty with 1 × loading buffer filling-in.90V, 20min, until all samples pressure
Being aligned；120V, 100min.
3) transferring film
The appropriately sized pvdf membrane of A, sanction, methyl alcohol activate the several seconds, standby in immersion transfer buffer.Remaining related equipment,
Such as sponge, filter paper, clamping plate etc., are also immersed in transfer buffer in advance.
B, the sponge for completing clamping plate lower floor and filter paper, test tube roll around bubble removing.
C, the careful PAGE glue that peels off on electrophoresis slide, are placed on filter paper.By on pvdf membrane rubber cover, then coated with filter
Paper and sponge, are rolled with test tube and remove bubble removing.
D, this sponge/filter paper/gel is put in electrophoretic apparatus according to producer's suggesting method, gel is towards negative electrode.
E, said apparatus are put in buffering liquid groove, and transfer buffer is filled to flood gel.
F, switch on power according to shown in producer, start transferring film, 100V, 2hr in ice chest.
4) immune response：
A, film, 5min is washed with TBST.
B, add and blockade liquid, steadily shake, room temperature 1hr.
C, TBST wash film, 5min.
D, add PDCD1 people source one anti-(being diluted with the TBST containing 3% skimmed milk power), room temperature 1hr or 4 DEG C of placement 12hr.
E, abandon one resist, TBST washes film, 15min × 3 time.
F, the two of addition horseradish peroxidase resist (mouse is anti-human) (being diluted with TBST), steadily shake, room temperature 2hr.
G, abandon two resist, TBST washes film, 15min × 3 time.
5) ECL exposure
It is ready to needed for exposure：Egative film, exposure, development, fixing solution etc..According to ECL product description, equal-volume mixes
Two components, cumulative volume 1ml are available for a film and use.In darkroom exposure 1～2min of exograph (after visual response light intensity and
Fixed), it is put into after washing in fixing solution, until egative film is fully transparent.Washing post-drying egative film.
The PD-1 albumen western testing result of the aim cell of each shRNA slow virus carrier transfection is shown in Fig. 2.
Then, the jamming effectiveness of three kinds of shRNA slow virus carriers, based on western testing result, is calculated, as a result as follows：
PDCD1-Homo-791 (target sequence SEQ ID NO：10：GCCACCATTGTCTTTCCTAGC) dry
Disturb efficiency：78%；
PDCD1-Homo-238 (target sequence SEQ ID NO：15：GCTAAACTGGTACCGCATGAG) dry
Disturb efficiency：8%；
PDCD1-Homo-694 (target sequence SEQ ID NO：5：GCCTGTGTTCTCTGTGGACTA) dry
Disturb efficiency：88%.
6) the quantity detection of PD1 positive cell
While the aim cell to the transfection of each shRNA slow virus carrier carries out flow cytometry analysis, PD1 positive cell is detected
Quantity, as a result see Fig. 3.
Thus, comprehensive western and flow cytomery result, it is known that high effect interference PD1 target spot be：
PD791：GCCACCATTGTCTTTCCTAGC(SEQ ID NO：10),
PD694：GCCTGTGTTCTCTGTGGACTA(SEQ ID NO：5),
And PD238 does not then have any interference effect to PD1.Two important PD1 disturbance target points are obtained in the present invention.
Embodiment 2
By taking the PD694 slow virus carrier that embodiment 1 is obtained as an example, using PD694 slow virus carrier, the interference for being carried
Fragment proceeds to immunocyte, prepares the immunocyte of PD-1 expression inhibiting, comprises the following steps that：
The separation of 1 PMNC
The cell that obtain will be separated from the 100ml peripheral blood of the positive liver cancer patient collection of 1 AFP, be transferred to 50ml from
(1800rpm, 10min) is centrifuged in heart pipe, supernatant is sucked, is taken the liquid after supernatant and is slowly added in lymphocyte separation medium Ficoll
On liquid (GE), volume ratio is 1:1, it is centrifuged (2000rpm, 20min)；
Then, the white flock cell for collecting interface adds PBS, and gently piping and druming is mixed, and is centrifuged (1500rpm, 10min)；
Next, repeated centrifugation washs totally 3 times；
Then, collect cell and mixing is gently blown and beaten, add basal medium 20ml piping and druming uniform, be transferred to 75cm2Blake bottle
In, in 37 DEG C, 5%CO2Incubator culture 2h.
2 lymphocytes and monocytic separation
Take out 75cm2Blake bottle, gently by 75cm2Blake bottle straighten vertical, draw 18ml nutrient solution in new 175cm2In bottle,
Activation culture for use in lymphocyte；
For former bottle (75cm2Blake bottle) in remaining 2ml raffinate, will be used for for monocyte being induced to differentiate into DC cell.
The culture of 3 lymphocytes
In the 175cm for cultivating lymphocyte2In bottle, 20ml CTL1 cell culture medium is added, after 24h, adds 20ml
CTL2 medium culture, then every 2～3d CTL3 culture medium equivalent fluid infusion, carries out active survival rate test and verifies whether to close
Lattice.
Cell culture medium is suctioned out, after PBS one time, is renewed fresh 15ml CTL3 culture medium, and adds 5*108PD694
Slow virus carrier mixes to blake bottle, changes liquid after 24 hours.
4. monocytic process
A, in the surplus former bottle (75cm for having 2ml raffinate2Blake bottle) in add 15ml DC culture medium to continue culture 4 days, per
Change liquid once within 2～3 days half, so as to monocyte is induced to differentiate into immature DC cell.
B, PEI infection protocol carries out antigen gene mRNA transfection：5th day, immature DC is collected with 50ml centrifuge tube thin
Born of the same parents (centrifugation 1500rpm, 10min), after discarding supernatant, lower confluent monolayer cells are transferred to 1.5ml centrifuge tube, are then directly added into
(i.e. AFP mRNA and PEI is according to mass ratio 1 for the reagent A of 100ul：The mixture obtained after 3 mixing) fully mix bottom
Portion's cell, covers centrifuge tube, is placed in cell culture incubator and is incubated 30 minutes, takes out turned upside down several times every 10 minutes.
Wherein, the preparation method of AFP mRNA refers to the applying date on May 5th, 2015, Application No. 201510225512.2
Patent application embodiment 1.
PEI (1 μ g/ μ l) Polysciences (CAT#23966-2 is configured to liquid storage) is to prepare to obtain according to following steps：
First, 80 DEG C or so dissolving PEI are heated to the sterilized water of endotoxin-free, are cooled to room temperature；Then, pH value is adjusted to 7.0,
Sterilization is filtered with 0.22 μm of filter, -20 DEG C after packing, are stored in, working solution can be saved backup at 4 DEG C.
Cell suspension is taken out with liquid-transfering gun and is added to blake bottle (75cm by c, taking-up centrifuge tube2) in, and DC culture medium is added,
Continue culture 3 days, to obtain ripe DC cell.
5. co-culture
After 8th day, by ripe DC cell with the aforementioned lymphocyte through activation culture according to volume ratio 1:20 mixing, use
CTL3 culture medium is co-cultured seven days, induces the generation of CTL cell and its homicidal wound cell.
After co-culturing seven days, flow cytometer detection is carried out to cell, is as a result found：The immunocyte group that the present embodiment is prepared
Not only contain substantial amounts of CTL cell in (PD694-CTL cell), and the panimmunity such as also NK and CIK cell is thin
Born of the same parents：CTL cell (CD8+, CD3+CD8+CD28+), CIK cell (CD3+CD56+), NK cell (CD3-CD56+),
I.e. immunocyte group not only has the powerful CTL cell for killing specific tumors cell, the also immunocyte such as CIK, NK.
Thus, inventor has found, the present invention is not only prepared for AFP Specific CTL Cells (PD694-CTL cell) in vitro,
While also NKT cell.These lymphocytes through the modification of the slow virus carrier of PD694 before antigenic stimulus is received,
Its PD1 has been suppressed up to more than 90%.
Embodiment 3：Clinical trial
The PD694-CTL cell that embodiment 1 is prepared feeds back patient, and observes feedback effect, specific as follows：
The positive liver cancer patient 20 of AFP is divided into 2 groups (A group 10 and B groups 10), wherein, for A group,
According to the method in embodiment 1 after the 14th day and the 15th day obtain enough CTL cells, feed back to patient, feed back every time
Cell concentration is 1～2*109；B group does not feed back cell.It is a course for the treatment of that blood sampling one-time continuous feed back 2 times.After 3 courses for the treatment of terminate,
Observed.
As a result find：After three courses for the treatment of of treatment, A group and B group have significantly difference.A group all patients are all felt after feedback
Body sense tired out mitigates, and appetite increases, pain relief, and body weight increase wherein has 8 patient's AFP indexs all to decline；
One patient has found primary million disappearance at 3 on liver by CT, and patient's index gets nowhere.And all patient's items diseases of B group
Shape does not all improve, and 10 patient's AFP indexs all increased., it is apparent that after feeding back PD694-CTL cell,
Patients symptomatic is relieved.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specific example ",
Or the description of " some examples " etc. means the specific features, structure, material or the feature bag that describe with reference to the embodiment or example
It is contained at least one embodiment or the example of the present invention.In this manual, the schematic representation to above-mentioned term not necessarily refers to
Be identical embodiment or example.And, the specific features of description, structure, material or feature can be at any one
Or combined in multiple embodiments or example in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Without departing from this
Multiple changes, modification, replacement and modification can be carried out to these embodiments in the case of the principle and objective of invention, the present invention's
Scope is limited by claim and its equivalent.
Claims (10)
1. one kind interferes fragment, the nucleotide sequence that its action target spot is as follows：
5′-GCCTGTGTTCTCTGTGGACTA-3′(SEQ ID NO：5).
2. interference fragment according to claim 1, it is characterised in that the interference fragment is by with following nucleotides sequences
The positive-sense strand of row and antisense strand composition：
Positive-sense strand：5′-GATCCGCCTGTGTTCTCTGTGGACTATTCAAGAGATAGTCCACAGAGAA
CACAGGCTTTTTTG-3′(SEQ ID NO：6),
Antisense strand：5′-AATTCAAAAAAGCCTGTGTTCTCTGTGGACTATCTCTTGAATAGTCCAC
AGAGAACACAGGCG-3′(SEQ ID NO：7).
3. a kind of method that PD-1 is expressed in suppression immunocyte, it is characterised in that include：
Interference fragment described in claim 1 or 2 is proceeded to the immunocyte.
4. method according to claim 3, it is characterised in that the interference fragment is turned using slow virus carrier system
Enter the immunocyte.
5. method according to claim 3, it is characterised in that the immunocyte is lymphocyte, preferably T cell,
B cell, NK cell, NKT cell, more preferably tumor-infiltrated T lymphocyte or CTL cell.
6. purposes of the interference fragment described in claim 1 or 2 in immunotherapy medicaments are prepared, includes PD-1 in the medicine
The immunocyte of expression inhibiting.
7. purposes according to claim 6, it is characterised in that the immunocyte of the PD-1 expression inhibiting be by with
Lower step is obtained：
Interference fragment described in claim 1 or 2 is proceeded to the immunocyte, to obtain the immunity of PD-1 expression inhibiting
Cell.
8. purposes according to claim 7, it is characterised in that the interference fragment is turned using slow virus carrier system
Enter the immunocyte.
9. purposes according to claim 7, it is characterised in that the immunocyte is lymphocyte, preferably T cell,
B cell, NK cell, NKT cell, more preferably tumor-infiltrated T lymphocyte or CTL cell.
10. a kind of immunotherapy medicaments, it is characterised in that the immunocyte comprising PD-1 expression inhibiting, the PD-1 table
The immunocyte for reaching suppression is to suppress PD-1 expression in immunocyte to obtain by the method described in any one of claim 3-5.
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