CN109097401A - A kind of preparation method of recombinant vector CAR-CD244-antiPSMA - Google Patents

A kind of preparation method of recombinant vector CAR-CD244-antiPSMA Download PDF

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CN109097401A
CN109097401A CN201810984124.6A CN201810984124A CN109097401A CN 109097401 A CN109097401 A CN 109097401A CN 201810984124 A CN201810984124 A CN 201810984124A CN 109097401 A CN109097401 A CN 109097401A
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antipsma
nk92mi
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顾雨春
尹乐
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National Health Chengnuo Biotechnology (Beijing) Co., Ltd.
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Beijing Promise Medical Science And Technology Co Ltd
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Abstract

The present invention relates to the preparation methods of recombinant vector CAR-CD244-antiPSMA a kind of, comprising the following steps: step 1, the primer by specific antibody encoding gene using end with BfuAI restriction enzyme enzyme recognition site carry out PCR amplification；The specific antibody coding gene sequence is the partial sequence of PSMA antibody, is sequence 3；Step 2 purifies the PCR product of step 1 respectively；Step 3, the PCR product for purifying step 2 carry out single endonuclease digestion in BfuAI restriction enzyme enzyme recognition site, and the cloning vector of the CD244 skeleton containing transformation is carried out single endonuclease digestion in BfuAI restriction enzyme enzyme recognition site simultaneously；Step 4 purifies product after digestion；Product after purification by antibody fragment and after containing the cloning vector molar ratio for the CD244 skeleton being transformed for 3:1 proportion, is attached using T4 ligase, is converted, coated plate, picking monoclonal extraction plasmid order-checking by step 5；Correct plasmid will be connected, will be named are as follows: recombinant vector CAR-CD244-antiPSMA.

Description

A kind of preparation method of recombinant vector CAR-CD244-antiPSMA
The application is divisional application, original application application No. is 201710209997.5, the applying date is March 31 in 2017 Day, it is entitled " a kind of CAR-NK cell and the preparation method and application thereof ".
Technical field
The present invention relates to field of biotechnology, specifically a kind of preparation side of recombinant vector CAR-CD244-antiPSMA Method.
Background technique
Cell biological treatment is the novel cancer therapies with self immune system, is a kind of with significant curative effect Treatment mode.It uses Protocols in Molecular Biology and cellular engineering technology, is exempted from biological agent to what is acquired from the patient Epidemic disease cell is cultivated and is expanded in vitro, then is fed back in patient body, so that the immune function of body itself is excited, enhances, Achieve the purpose that monitoring and kills sick cell or repair damaged cell.
Natural killer cells (natural killer cell, NK) belongs to granular lymphocytic, and being that body is important is immunized Cell.NK cell origin is thought as two in marrow lymphoid stem cells, differentiation, development depend on marrow or thymus microenvironment, It is distributed mainly on peripheral blood and spleen, also has a small amount of presence in lymph node and its hetero-organization.Expression specificity is not anti-for NK cell Former identification receptor is the third quasi-lymphocyte different from T, bone-marrow-derived lymphocyte.Compared with other lymphocytes, NK cell is killed Wound activity is limited without MHC, is not necessarily to antigen presensitization, because of referred to herein as Nk Cell Activity.NK cell cytosol is abundant, containing larger Azurophilic granule, and the content of azurophilic granule and the killing activity of NK cell are positively correlated, therefore NK cytosis is thin in target Lethal effect occurs early after born of the same parents, 1 hour in vitro, internal 4 hours i.e. visible lethal effect.The target cell of NK cell mainly has Certain tumour cells (including part cell line), virus infected cell etc., therefore NK cell can the certain tumour cells of direct killing And virus infected cell.This means that NK cell is body is antitumor, it is important to play in viral infection resisting and immunological regulation Effect.
CD244 is the human protein encoded by CD244 gene, also known as NK cell receptor 2B4, is that a NK cell is important Surface receptor.NK cell passes through CD244 receptor activation cytotoxicity function.
CAR means Chimeric antigen receptor (Chimeric Antigen Receptor), when CAR structure is related to tumour anti- Original antibody amalgamation and expression can assign the energy that NK cell-specific identifies corresponding tumour cell in the cell membrane surface of NK cell Power, NK cell at this time are CAR-NK cell.One complete CAR structure generally includes film signaling zone, antibody district (usually From the scFV section of corresponding monoclonal antibody), extracellular school sequence, transmembrane region, intracellular signal area.The NK crossed by gene modification Cell (CAR-NK) can generate the stronger killing functions of immunocytes of specificity to the cell with its antigen being directed to, so as to Achieve the effect that treatments such as tumours.
Slow virus carrier refers to a kind of viral vectors in the source human immunodeficiency virus-1 (HIV-l), can will be outer Source gene is effectively integrated on host chromosome, is that foreign gene is normal to achieve the effect that persistence expresses aim sequence With one of carrier format.Its basic process is exactly to carry the slow virus carrier of foreign gene in slow virus packaging plasmid, cell Under the auxiliary of system, becomes infectious virion by virus packaging, by infection cell or living tissue, realize external source Gene is expressed in a cell or in a living tissue.Slow virus carrier can effectively infect neuronal cell in terms of infection ability, swell A plurality of types of cells such as oncocyte, stem cell, cardiac muscle cell.More convenient mesh can be quickly realized using slow virus carrier Gene it is long-term, stablize expression.
Currently, not yet establishing a kind of method prepared by effective CAR-NK cell and its answering in terms of immunotherapy of tumors Use precedent.
Summary of the invention
In view of the deficiencies in the prior art, the purpose of the present invention is to provide a kind of CAR-NK cell and its preparation sides Method and application.
The purpose of the present invention and technical problems to be solved are: transformation source of people CD244 sequence, enable thin in NK etc. Cellular surface expression specificity albumen, which can identify specific tumors cell, while can activate NK cell, be allowed to With the killing ability to specific tumors cell.It include that the slow virus carrier of the CD244 sequence of transformation can be coated with slow virus, It can make the CD244 protein sequence of NK cell expression transformation after slow-virus infection NK cell.
To achieve the above objectives, the technical solution adopted by the present invention is that:
The present invention provides a kind of preparation method of CAR-NK cell, includes the following steps:
Sl, specific antibody (the specific antibody CD19 antibody or PSMA antibody of such as tumour cell target antigen) is encoded Gene (coupling part of chain moiety, heavy chain and light chain comprising specific antibody and heavy chain moiety) is connected to modified CAR gene is obtained in the cloning site area of CAR skeleton,
The modified CAR skeleton includes film signaling zone on sequentially connected CD244, cloning site area, CD244 extracellular Hinge area, CD244 transmembrane segment area and CD244 intracellular part area,
S2, the CAR gene is passed through into slow-virus infection NK cell, obtains expressing the CAR albumen (the i.e. described CAR base The expression albumen of cause, such as the CAR-CD244-antiCD19 protein structure and CAR-CD244-antiPSMA albumen in embodiment 4 Structure) NK cell (such as NK92MI cell), i.e., the described CAR-NK cell.
In the preparation method of above-mentioned CAR-NK cell, the DNA sequence dna of film signaling zone such as sequence on the CD244 Shown in the 1st to the 63rd in 1；
And/or the DNA sequence dna in the cloning site area such as the 64th in sequence 1 is to shown in the 95th；
And/or the extracellular hinge area of the CD244, CD244 transmembrane segment area and the DNA sequence dna in CD244 intracellular part area are such as Shown in the 96th to the 560th in sequence 1.
In the preparation method of above-mentioned CAR-NK cell, the DNA sequence dna such as sequence of the modified CAR skeleton Shown in 1.
In the preparation method of above-mentioned CAR-NK cell, the step S2 is carried out according to the method included the following steps:
S21, the CAR gene is connected into acquisition recombined lentivirus vector A on the cloning site of slow virus carrier, will recombinated Slow virus carrier A transfection aim cell (such as 293T cell) is cultivated, and slow virus is obtained；
S22, by slow-virus infection NK cell, obtain the CAR-NK cell.
In the preparation method of above-mentioned CAR-NK cell, in step S21, the slow virus carrier is Plenti slow virus load Body；
The transfection is carried out using slow virus packaging plasmid；
The slow virus packaging plasmid is specially psPAX2 and PMD2.G, when transfection, according to by the slow virus carrier, PsPAX2 and PMD2.G is that 5ug:3.2ug:l.8ug--7.5ug:4.8ug:2.7ug transfects 3 × I0 than ratio with quality6A mesh Cell；
In step S22, described by slow-virus infection NK cell includes that the slow virus of concentration is added in complete medium, The step of being mixed with NK cell.
In the preparation method of above-mentioned CAR-NK cell, in step S22, it is described by slow-virus infection NK cell further include plus The step of entering polybrene；The final concentration of 4ug/mL of the addition of the polybrene；
The slow virus of concentration is after being cultivated recombined lentivirus vector A transfection aim cell, by the supernatant of culture After being centrifuged 1 hour under the conditions of 20000g, takes slow virus to be suspended with DPBS buffer and obtain.
It is following any one of (1) or (2) the present invention also provides a kind of DNA molecular:
It (1) include film signaling zone on sequentially connected CD244, cloning site area, the extracellular hinge area of CD244, CD244 cross-film Part area and CD244 intracellular part area；
It (2) is to be inserted into specific antibody (such as tumour cell target antigen in the cloning site area of (1) described DNA molecular Specific antibody) DNA molecular that is formed after encoding gene.
In above-mentioned DNA molecular, the 1st on the CD244 in the DNA sequence dna of film signaling zone such as sequence 1 is extremely Shown in 63rd；
And/or the DNA sequence dna in the cloning site area such as the 64th in sequence 1 is to shown in the 95th；
And/or the extracellular hinge area of the CD244, CD244 transmembrane segment area and the DNA sequence dna in CD244 intracellular part area are such as Shown in the 96th to the 560th in sequence 1；
The sequence of the DNA molecular of (1) concretely sequence shown in sequence 1.
Recombinant vector (such as recombinant expression carrier, recombined lentivirus vector), again of the present invention protection containing the DNA molecular Group cell (such as immunocyte NK cell or T cell or 293T cell) or recombinant cell lines.
The present invention protects the DNA molecular, the recombinant vector, recombinant cell or recombinant cell lines, any above method CAR-NK cell is prepared in preparation oncotherapy (as inhibited tumour growth or killing tumor cell) drug (such as tumour immunity Treat cell), the application in reagent or kit.
Contain following liquid in the preparation tumor therapeutic agent, reagent or kit: being 5 × 10 containing concentration6It is a described CAR-NK cell/100ul physiological saline.
The invention has the following beneficial effects:
The present invention is natural killer cells (NK cell), NK for constructing the cell material of novel tumor immunization therapy cell Cell immunogenicity is low, can carry out allosome feedback, solve the problems, such as cell origin compared to T cell；
Tumor vaccine prepared by the present invention can induce the target cell lysis of antigentic specificity；
Modified CAR skeleton of the present invention is equally applicable to other immunocytes；Through preparation method institute of the invention The NK cell of modification can generate the stronger cell killing of specificity to such as tumour cell of the cell with its antigen being directed to and make With so as to achieve the effect that the treatments such as tumour.
Immunotherapy of tumors cell prepared by the present invention also can be applied to different in addition to it can be applied to cancer patient Body is fed back.Therefore, for the immunotherapy of tumors cell prepared by the present invention compared with existing CAR-T, use scope is wider, preparation Cost is lower.
Detailed description of the invention
The present invention has following attached drawing:
Fig. 1 is the modified CD244 skeleton schematic diagram for being connected to specific antibody encoding gene.
Fig. 2 is recombined lentivirus vector Plenti-CAR-CD244-antiCD19 schematic diagram.
Fig. 3 is expression figure of the CD244-antiCD19 and CD244-antiPSMA in NK92MI cell.
Fig. 4 is CAR structure representation result in Flow cytometry NK cell, wherein ordinate is to count, and abscissa is The green fluorescence intensity (logarithm) of CD19-FITC (GRN-Hlog).
Fig. 5 is killing experiments of the NK92MI-CD244-antiCD19 to Daudi tumour cell.
Fig. 6 is killing experiments of the NK92MI-CD244-antiPSMA to PC3 tumour cell.
Fig. 7 is IFN γ Interferon level detection after NK92MI-CD244-antiCD19 is incubated for Daudi tumour cell 20h.
Fig. 8 is IFN γ Interferon level detection after NK92MI-CD244-antiPSMA is incubated for PC3 tumour cell 20h.
Fig. 9 is that the tumour growth situation result after living body fluorescent imaging is carried out to mouse.
Specific embodiment
Below in conjunction with attached drawing, invention is further described in detail.
Embodiment 1, recombinant vector CAR-CD244-antiCD19 and recombinant vector CAR-CD244-antiPSMA preparation
1. the primer by specific antibody encoding gene using end with BfuAI restriction enzyme enzyme recognition site carries out PCR amplification；
The specific antibody coding gene sequence used in the present embodiment is respectively CD19 antibody (antiCD19) The partial sequence (sequence 3) of partial sequence (sequence 2) and PSMA antibody (antiPSMA)；
2. the PCR product of step 1 is purified respectively；
3. the PCR product that step 2 is purified carries out single endonuclease digestion (BfuAI), and simultaneously by the CD244 skeleton containing transformation Cloning vector carries out single endonuclease digestion (BfuA I)；
4. product after digestion is purified；
5. being 3:1 by the cloning vector molar ratio of CD244 skeleton of the product after purification by antibody fragment and containing transformation It after proportion, is attached, converted using T4 ligase, coated plate, picking monoclonal extract plasmid order-checking；Correct plasmid will be connected (being connected into antiCD19 or antiPSMA in the cloning site area of the CD244 skeleton of the transformation) is respectively designated as: recombination Support C AR-CD244-antiCD19 and recombinant vector CAR-CD244-antiPSMA.
The CD244 frame sequence of the transformation is as shown in sequence 1, wherein the 1st in sequence 1 is extremely 63rd shown sequence is the DNA sequence dna of film signaling zone on CD244；The 64th to the 95th shown sequence in sequence 1 It is classified as the DNA sequence dna in cloning site area；The 96th to the 560th shown sequence in sequence 1 is the extracellular hinge of CD244 Area, CD244 transmembrane segment area and CD244 intracellular part area DNA sequence dna (extracellular hinge area be included in transmembrane segment area with it is intracellular Part area) in；The 561st to the 575th shown sequence in sequence 1 is DDDDK-tag label；Sequence 1 In the 576th to the 578th shown sequence be terminator codon.
The CD244 skeleton schematic diagram of the transformation of specific antibody encoding gene is connected as shown in Figure 1: in Fig. 1, CD244leader represents film signaling zone on CD244, VLThe chain moiety of specific antibody is represented, GSlinker represents specificity The coupling part of heavy chain of antibody and light chain, VHThe heavy chain moiety of specific antibody is represented, CD244 represents the extracellular hinge of CD244 Area, transmembrane segment area and intracellular part area, DDDDK represent DDDDK-tag label.
The preparation of embodiment 2, recombined lentivirus vector
1. the recombinant vector CAR-CD244-antiCD19 and recombinant vector CAR-CD244- that are prepared respectively with embodiment 1 AntiPSMA is expanded as template using the primer PCR with BamHI and XbaI restriction enzyme digestion sites sequence；
2. a pair PCR product is purified respectively, digestion (BamHI and Xba I), purifying；
3. it is (entitled that the product of step 2 is connected into Plenti slow virus carrier respectively respectively using T4 ligase PLentiCMV/TO eGFP Puro (w159-1) is purchased from addgene, network address: http://www.addgene.org/ 17481/) between the site BamHI and XbaI；
4. conversion, coated plate, picking monoclonal extract plasmid order-checking, correct recombined lentivirus vector will be sequenced and name respectively For recombined lentivirus vector Plenti-CAR-CD244-antiCD19 and Plenti-CAR-CD244-antiPSMA.
The schematic diagram of recombined lentivirus vector Plenti-CAR-CD244-antiCD19 is as shown in Figure 2, wherein CMV- Promoter represents CMV promoter, and CD244peptide is film signaling zone on CD244, and antiCD19 is CD19 antibody, CD244 For the extracellular hinge area of CD244, transmembrane segment area and intracellular part area, DDDDK is DDDDK-tag label, and WPRE is that function is similar In the region of the 3'UTR of polyA.
The preparation and concentration of embodiment 3, slow virus
1, the preparation of slow virus
Recombined lentivirus vector Plenti-CAR-CD244-antiCD19 and the psPAX2 (purchase prepared using embodiment 2 (it is purchased from addgene, network address is from addgene, network address https: //www.addgene.org/12260/), pMD2.G Https: //www.addgene.org/12259/) carrier is 5ug:3.2ug:1.8ug transfection 293T cell (about 3 in mass ratio ×106A 293T cell is laid on 10cm ware), fresh culture (DMEM culture medium adds 10% fetal calf serum) is changed to after 8h, Later every collecting a supernatant for 24 hours and being added fresh culture (DMEM culture medium adds 10% fetal calf serum), 3 are collected altogether It is secondary, obtain slow virus supernatant A.
Using embodiment 2 prepare by recombined lentivirus vector Plenti-CAR-CD244-antiPSMA and psPAX2, PMD2.G carrier is 5ug:3.2ug:1.8ug transfection 293T cell (about 3 × 10 in mass ratio6A 293T cell is laid on 10cm Ware), fresh culture (DMEM culture medium adds 10% fetal calf serum) is changed to after 8h, later every collecting a supernatant for 24 hours And fresh culture (DMEM culture medium adds 10% fetal calf serum) is added, it collects 3 times altogether, obtains slow virus supernatant B.
2, the concentration of slow virus
The slow virus supernatant A and B that step 1 is obtained respectively is packed into high speed centrifugation pipe by 30ml/ pipe, uses supercentrifuge Centrifugation, 20000g are centrifuged 1 hour, collect slow virus on tube wall, are suspended using 100ul DPBS buffer, obtain concentration respectively Slow virus A and B, -80 DEG C of preservations.
Embodiment 4, the slow-virus infection NK cell preparation CAR-NK cell of concentration and detection
1, the slow-virus infection NK cell being concentrated
1ml complete medium is added, with 10 in the slow virus A and B of the concentration respectively prepared by embodiment 35A NK92MI Cell (is purchased from ATCC, network address: https: //www.atcc.org/) mixing and (polybrene of final concentration 4ug/ml is added Polybrene), recombinant cell NK92MI-CD244-antiCD19 and recombinant cell NK92MI-CD244-antiPSMA are obtained (recombinant cell obtained herein is cell mixing, and wherein CAR-NK cell refers to the recombinant cell NK92MI-CD244- of NK92MI AntiCD19 and NK92MI-CD244-antiPSMA).
2, detection of expression of the CAR structure in NK cell
By the 293T transfection carrier that step 1 obtains recombinant cell NK92MI-CD244-antiCD19 and embodiment 3 obtains The cell of Plenti-CAR-CD244-antiCD19 carries out Western blot and detects surface antibody expression: with NK92MI Cell after transfecting Plenti empty carrier is control, transfects Plenti empty carrier respectively at NK92MI using CD19-FITC antigen After cell, recombinant cell NK92MI-CD244-antiCD19 afterwards is incubated for, Western blot detection is carried out, as a result such as Shown in Fig. 3.
Fig. 3's the result shows that, NK92MI-CD244-antiCD19 and 293T transfection carrier Plenti-CAR-CD244- The cell of antiCD19 being capable of specifically expressing CAR-CD244-antiCD19 protein structure.
The 293T transfection carrier Plenti-CAR- that recombinant cell NK92MI-CD244-antiPSMA and embodiment 3 obtain The detection of expression of the cell of CD244-antiPSMA carries out according to the method described above, as a result, NK92MI-CD244-antiPSMA and The cell of 293T transfection carrier Plenti-CAR-CD244-antiPSMA being capable of specific expressed CAR-CD244-antiPSMA egg White structure.
3, in Flow cytometry CAR-NK cell CAR protein structure expression
Concrete operations are carried out by streaming antigenic label operation instruction, specific as follows:
Cell to be detected (the recombinant cell NK92MI-CD244-antiCD19 in step 2) is taken about 105A cell centrifugation After remove supernatant；
Using 200ul DPBS suspension cell to be detected, streaming antigenic label is added, mixes gently, in 37 DEG C of shaking tables (100 turns) are incubated for 30 minutes, and supernatant is removed in centrifugation；
Using 500ul complete medium suspension cell, 37 DEG C incubator culture 5 minutes, supernatant is abandoned in centrifugation；
After cell is resuspended using 200ul DPBS, upper machine testing.
As a result: as shown in figure 4, Flow cytometry recombinant cell NK92MI-CD244-antiCD19 cell surface CAR The expression of protein structure, darker non-flanged solid line grey parts are without any modification NK92MI cell controls, and shallower has reality Line edge grey parts are that recombinant cell NK92MI-CD244-antiCD19 is incubated for CD19-FITC antigen, and displacement illustrates that recombination is thin Born of the same parents' NK92MI-CD244-antiCD19 cell membrane surface has CD19 antibody expression；Boundary line is corresponding abscissa fluorescence signal position Set corresponding cell count.
The killing experiments of embodiment 5, CAR-NK cells against tumor cells
Take 104A NK92MI-CD244-antiCD19 cell and 104A bone-marrow-derived lymphocyte tumor (Daudi) cell incubation, with NK92MI is control, is denoted as 1:1；
5,000 NK92MI-CD244-antiCD1 cells and 104A Daudi is incubated for, and is control with NK92MI, is denoted as 0.5:1；
2,500 NK92MI-CD244-antiCD19 cells and 104A Daudi is incubated for, and is control with NK92MI, is denoted as 0.25:1；
1,250 NK92MI-CD244-antiCD19 cell and 104A Daudi is incubated for, and is control with NK92MI, is denoted as 0.125:1；
It is control with NK92MI by NK92MI-CD244-antiPSMA and prostate cancer (PC3) cell incubation, ratio is pressed The above method carries out.
If three groups of parallel controls, detected after being incubated for 20 hours using CellTox-Green (promega G8471) kit Cell viability (universal method), drawing.
As a result as shown in Figure 5 and Figure 6, wherein Diamond spot is the oncolysis under the conditions of unmodified NK92MI cell Degree；Square points are that (NK92MI-antiCD19 represents NK92MI-CD244- for the NK92MI cell modified through 4 method of embodiment AntiCD19, NK92MI-antiPSMA represent NK92MI-CD244-antiPSMA) under the conditions of tumor lysis degree.As a result it demonstrate,proves The NK cells against tumor cells of the NK cell of bright recombinant C D244 modified, i.e. CAR-NK recombinant cell compared to unmodified mistake Fragmentation effect is more preferable.
IFN γ Interferon level detection after embodiment 6, CAR-NK cell incubation Daudi, PC3 cell 20h
Take 105A NK92MI-CD244-antiCD19 and 105A Daudi cell is incubated for 20h (note in 500ul culture medium For NK92MI-antiCD19+Daudi)；It is control (being denoted as NK92MI+Daudi) with unmodified NK92MI cell；
Take 105A NK92MI-CD244-antiPSMA cell and 105A PC3 cell is incubated for 20h in 500ul culture medium (being denoted as NK92MI-antiPSMA+PC3)；It is control (being denoted as NK92MI+PC3) with unmodified NK92MI cell；
By come in the cell centrifuging and taking after incubation, row IFN γ Interferon level is detected, and specific method is used by kit Illustrate to carry out (different kit methods are slightly different).
As a result as shown in Figure 7 and Figure 8, wherein NK92MI be unmodified NK92MI cell be individually incubated for as a result, NK92MI-antiCD19 is that NK92MI-CD244-antiCD19 is individually incubated for as a result, NK92MI-antiPSMA is The result that NK92MI-CD244-antiPSMA is individually incubated for.
The result shows that: NK cell, that is, CAR-NK cell NK92MI-CD244-antiCD19 of recombinant C D244 modified and NK92MI-CD244-antiPSMA can discharge more IFN γs after being incubated for corresponding tumour cell, illustrate CAR-NK activity ratio Unmodified common NK cell activity is stronger, higher to tumor cytotoxicity effect.
Embodiment 7, experimental animal tumor model experiment in vivo
The nude mice (Nude Mouse) of health is randomly divided into 3 groups for totally 9, every group 3,3 groups are respectively blank control group, NK cell controls group, CAR-NK groups of cells.5 × 10 are injected near the oxter of all mouse shirtfronts6It is a to have GFP fluorescent protein labeling PC3 tumour cell/only, and normally feed 14 days.After 14 days, living body fluorescent imaging, record are carried out to all groups of other mouse The growth situation of mouse interior tumor.After fluorescence imaging, respectively to each group mouse PC3 tumor cell injection point nearby respectively into The following administration of row:
Give blank control group mouse injecting normal saline 100ul；
Common NK92MI cell suspending liquid 100ul is injected to NK cell controls group mouse, sum is 5 × 106A NK cell/ 100ul physiological saline；
To CAR-NK (NK92MI- made of embodiment 4 of the CAR-NK groups of cells mouse injection needle for PC3 tumour cell CD244-antiPSMA) (concentration is 5 × 10 to cell suspending liquid6A CAR-NK (NK92MI-CD244-antiPSMA) cell/ 100ul physiological saline).
After injection and normal feed 7 days carries out living body fluorescent imagings, record to all groups of other mouse respectively later Mouse interior tumor situation.
As a result as shown in figure 9, to be respectively as follows: blank control group, NK cell controls group, CAR-NK thin for the column of left, center, right three in Fig. 9 Born of the same parents' group, the first behavior inject after as a result, the second behavior normally feed 7 days after as a result, Fig. 9 the results show that compared to Blank control group and NK cell controls group, the intracorporal mean tumour volume of CAR-NK groups of cells mouse is significantly smaller, tumour growth Obviously it is inhibited.
The content being not described in detail in this specification belongs to the prior art well known to professional and technical personnel in the field.

Claims (2)

1. a kind of preparation method of recombinant vector CAR-CD244-antiPSMA, which comprises the following steps:
Step 1, the primer by specific antibody encoding gene using end with BfuAI restriction enzyme enzyme recognition site carry out PCR amplification；
The specific antibody coding gene sequence is the partial sequence of PSMA antibody, is sequence 3；
Step 2 purifies the PCR product of step 1 respectively；
Step 3, the PCR product for purifying step 2 carry out single endonuclease digestion in BfuAI restriction enzyme enzyme recognition site, and simultaneously will The cloning vector of CD244 skeleton containing transformation carries out single endonuclease digestion in BfuAI restriction enzyme enzyme recognition site；
Step 4 purifies product after digestion；
Product after purification by antibody fragment and is contained the cloning vector molar ratio for the CD244 skeleton being transformed for 3:1 by step 5 It after proportion, is attached, converted using T4 ligase, coated plate, picking monoclonal extract plasmid order-checking；It will be in the transformation The cloning site area of CD244 skeleton is connected into antiPSMA, obtained plasmid name are as follows: recombinant vector CAR-CD244- antiPSMA。
2. the preparation method of recombinant vector CAR-CD244-antiPSMA as described in claim 1, which is characterized in that described to change The CD244 frame sequence made is as shown in sequence 1, wherein the 1st to the 63rd shown sequence in sequence 1 For the DNA sequence dna of film signaling zone on CD244；The 64th to the 95th shown sequence in sequence 1 is cloning site area DNA sequence dna；The 96th to the 560th shown sequence in sequence 1 is the extracellular hinge area of CD244, CD244 cross-film portion The DNA sequence dna of subregion and CD244 intracellular part area；The 561st to the 575th shown sequence in sequence 1 be DDDDK-tag label；The 576th to the 578th shown sequence in sequence 1 is terminator codon.
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