CN105925544A

CN105925544A - Preparation method and application of 4-1BB-containing lentivirus

Info

Publication number: CN105925544A
Application number: CN201610482530.3A
Authority: CN
Inventors: 董坚; 杨益; 刘玉芝; 何群; 周棠怡; 梁福蓉
Original assignee: Wuhan Think Medical Technology Co Ltd
Current assignee: Wuhan Think Medical Technology Co Ltd
Priority date: 2016-06-27
Filing date: 2016-06-27
Publication date: 2016-09-07

Abstract

The invention provides a preparation method and application of 4-1BB-containing lentivirus. According to the lentivirus prepared by the preparation method for the lentivirus, CD28 can be not required to be added; only a small amount of CD3 is directly added to a culture medium to serve as a first T-cell activating signal before transfection, so that a process of preparing Car-T cells is reduced, and the cost and pollution risk are reduced; T-cells are stimulated to be further activated through recombinant VSVG (Vesicular Stomatitis Virus Glycoprotein), so that the contact probability of a virus and cells is improved to effectively improve infection efficiency.

Description

A kind of slow virus preparation method and application comprising 4-1BB

Technical field

The invention belongs to medical biotechnology field, be specifically related to a kind of slow virus preparation side comprising 4-1BB Method and application.

Background technology

Slow virus (Lentivirus) carrier is to develop based on HIV-1 (human immune deficiency I type virus) The gene therapy vector got up.Distinguishing general retroviral vector, it is thin to somatoblast and nondividing Born of the same parents are respectively provided with infection ability.The research and development of slow virus carrier obtains quickly, research the most deep.This load Exogenous gene can be incorporated on host chromosome by body effectively, thus reach persistency and express.Infecting Ability aspect can effectively infect neuronal cell, hepatocyte, myocardial cell, tumor cell, endotheliocyte, Polytype cell such as stem cell, thus reach good gene therapy effect, carry out in the U.S. Clinical research, effect is ideal, therefore have broad application prospects.

Lentiviral contains the hereditary information required for packaging, transfection, stable integration.Slow virus Packaging plasmid can provide all of transcribe and pack RNA to restructuring Pseudovirion vector required for all auxiliary Help albumen.For producing the virion of high titre, need to utilize expression vector and packaging plasmid cotransfection simultaneously Cell, carries out the packaging of virus in cell, and packaged pseudovirion is secreted into extracellular culture medium In, after centrifuging and taking obtains supernatant, it being used directly for the infection of host cell, genes of interest enters into host After cell, through reverse transcription, it is incorporated into genome, thus high-caliber expression effect molecule.

As it is shown in figure 1, slow virus can be as the carrier of the exogenous gene in the treatment of CART lymphocyte, will Exogenous gene CAR imports in T lymphocyte, thus T lymphocyte is carried out genomic modification, with Time play the effect to T lymphocyte activation.The T cell of Chimeric antigen receptor (CAR) genetic modification is So that the fusion base of the chimeric molecule of single-chain antibody-costimulatory molecules-ITAM can be encoded The T cell of a kind of genetic modification produced because modifying T cell, have tumor antigen identification specificity strong, Affinity MHC high, non-is restricted and can expand the most in a large number.

As prior art immediate with the application, the patent of Publication No. CN201510279570.3 is open A kind of slow virus with high-efficiency transfection ability and biologic activity for preparing CART cell, will The correlated series of CD3 and CD28 is used for the packaging of slow virus with VSVG after recombinating, by first letter of CD3 Number, the secondary signal of CD28, individually carry out common activating T cell, simultaneously in sense with VSVG restructuring Not using any antibody to activate T cell before dye T cell, this activation method has certain risk and not Stability.

Summary of the invention

In order to solve the problems referred to above that prior art exists, the invention provides a kind of slow disease comprising 4-1BB Poison preparation method and application.The slow virus prepared by described slow virus preparation method, it may not be necessary to add CD28, only before transfection, directly adds a small amount of CD3 in culture medium and believes as t cell activation first Number, reduce program prepared by Car-T cell, reduce cost and pollution risk；By restructuring VSVG, thorn Sharp T cell activates further, increases virus with cells contacting probability to be effectively improved efficiency of infection.

The technical solution adopted in the present invention is:

A kind of slow virus preparation method comprising 4-1BB, comprises the following steps:

A, the structure of slow virus carrier plasmid 881-3-SJ19:

A1, CD19CAR gene is carried out PCR amplification: with full genome synthesis CD19CAR gene as mould Plate, carries out PCR amplification, obtains CD19CAR amplified production；

A2, gel purify CD19CAR amplified production: use gel to purify in pcr amplification product The cDNA of CD19CAR；

A3, use CD19CAR gene transfecting cell: by the cDNA of the CD19CAR that obtains in step A2, 18-T Vector and Solution I mix homogeneously, preserve 16 hours at 16 DEG C；It is subsequently adding Trans5 α In competent cell, in ice, keep 30min successively, be then heated to 42 DEG C and keep 45s, then protect in ice Hold 1min, be subsequently adding SOC/LB culture medium, 37 DEG C of shaken cultivation 60 minutes, centrifugal after cultivation, After abandoning part supernatant, remaining liq is coated on the Agar Plating containing Amp cultivation；

A4, the extraction plasmid containing CD19CAR gene: picking 881-3-SJ19 from Agar Plating Plasmid；

The transformation of B, VSVG plasmid:

B1,4-1BB gene is carried out PCR amplification: with the genetic fragment of full genome synthesis 4-1BB as mould Plate, carries out PCR amplification, obtains 4-1BB amplified production；

B2, VSVG gene is carried out PCR amplification: with the genetic fragment of full genome synthesis VSVG as mould Plate, carries out PCR amplification, obtains VSVG amplified production；

B3, acquisition 4-1BB-VSVG recombination fragment: by described 4-1BB amplified production and VSVG Amplified production carries out enzyme action, and reclaims digestion products and be attached reaction, obtains connecting product；

B4, with 4-1BB-VSVG recombination fragment transfection cell: the connection product that step B3 is obtained Add in TOP10 competent cell, in ice, keep 30min after mix homogeneously successively, be then heated to 42 DEG C Keep 45s, then in ice, keep 1min, be subsequently adding SOC/LB culture medium, 37 DEG C of shaken cultivation 60 Minute, centrifugal after cultivation, after abandoning part supernatant, remaining liq is coated on Agar Plating training Support；

B5, the extraction of transformation VSVG plasmid: extract transformation with alkaline lysis or plasmid extraction test kit VSVG plasmid, named 4-1BB-VSVG plasmid；

C, the preparation of slow virus:

C1, preparation BES solution: by the BES of NaCl, 5ml 0.5M of 10ml 1.4M, 500 μ L 150mM Na₂HPO₄With the NaOH mix homogeneously of 1100 μ L 1M, add distilled water and be settled to 50ml, adjust PH To 6.95, preserve after filtration, be BES solution；

C2, preparation transfection reagent: 881-3-SJ19 plasmid that step A4 is obtained, NRF plasmid, step 4-1BB-VSVG plasmid, 1M CaCl2 and the distilled water that B5 obtains, after mix homogeneously, is added dropwise over step The BES solution that rapid C1 obtains, after dropping, stands 30min；Obtain transfection reagent；

C3, cell transfecting and cultivation: recovery 293T cell is also cultivated, when described 293T cell density Reach bottom culture dish to transfect when 70%, thin to described 293T by the DMEM culture medium without serum Born of the same parents carry out changing liquid；After changing liquid, it is added dropwise over the transfection reagent that step C2 obtains, after mix homogeneously, by described 293T cell puts into incubator, stands 2 hours, static after drip hyclone, continue to cultivate 8 hours, Then use the DMEM containing 10wt% hyclone to change liquid, continue to cultivate 48 hours；

C4, collection virus: after step C3 completes, collect supernatant, after centrifugal filtration, toxogen of i.e. falling ill Liquid.

The slow virus prepared by described slow virus preparation method, it may not be necessary to add CD28, only in transfection Before, the CD3 that directly interpolation is a small amount of in culture medium, as t cell activation the first signal, reduces Car-T thin Program prepared by born of the same parents, reduces cost and pollution risk；By restructuring VSVG, T cell is stimulated to live further Change, increase virus with cells contacting probability to be effectively improved efficiency of infection.

According to one embodiment of present invention, the operation of gel purification CD19CAR amplified production includes following Step:

1. under uviol lamp, cut out the agarose gel containing target DNA, shred blob of viscose.

2. weigh blob of viscose weight, calculate blob of viscose volume, calculate with 1mg=1 μ L, in blob of viscose, add Buffer GM (3 gelinite accumulated amounts).

3. uniformly the rear 37 DEG C of water-baths of mixing are dissolved, and now should be interrupted vibration mixing, make blob of viscose fully dissolve (about 5-10 minute).

4. after gel is completely dissolved, observe the color of sol solutions, if sol solutions color from yellow becomes orange Color or pink colour, add 3M sodium acetate solution (pH5.2) 10 μ l in above-mentioned blob of viscose lysate, uniformly mix It is bonded to solution and recovers yellow.When separating the DNA fragmentation less than 400bp, should add in this solution The isopropanol of final concentration of 20%.

5. the Spin Column in test kit is placed on Collection Tube.

6. being transferred in Spin Column by the solution of aforesaid operations step 4,12,000rpm are centrifuged 1 point Clock, abandons filtrate.

7. being added in Spin Column by the Buffer WB of 700 μ l, room temperature 12,000rpm is centrifuged 30 Second, abandon filtrate.

8. add the Buffer WB of 700 μ l, Spin Column be placed on Collection Tube, Room temperature 12,000rpm is centrifuged 1 minute.

9. Spin Column is placed on the centrifuge tube of new 1.5ml, in the central authorities of Spin Column film Place adds 30 μ l sterile purified waters or Elution Buffer (is heated to during 60 DEG C of uses being conducive to improving eluting Efficiency.) room temperature stand 1 minute.Room temperature 12000rpm is centrifuged 1 minute eluted dna.

In step A1, the cumulative volume of described PCR reaction system is 50 μ L: include the Ex Taq of 0.25 μ L Enzyme, the Ex Taq buffer of 5 μ L, the dNTP of 4 μ L, the template of 2 μ L and 4 μ L primers, remaining Amount is aquesterilisa, and the concentration of the dNTP used is 2.5mM, and the concentration of template is 100ng/ μ L, primer Concentration be 10uM.

It is to say, described dNTP, template and primer, it is the concentration before interpolation.

In step A1, the PCR amplification program of use is: carrying out 25 circulations, the most each circulation is successively Comprising the following steps: denaturation 10s at a temperature of 98 DEG C, anneal at a temperature of 50 DEG C 30s；72 DEG C of temperature 2min is extended under degree；After completing 25 circulations, i.e. complete PCR amplification program.

After step A4 completes, also include that A5, enzyme action detect: carry out enzyme action step and coupled reaction successively Detect the sequence of described 881-3-SJ19 plasmid；The enzyme action system used is: the 881-3-SJ19 plasmid of 2 μ L, The buffer of 1 μ L, the XhoI enzyme of 0.5 μ L and the ddH of 6.5 μ L₂O；The linked system used is: 2 The slow virus carrier of μ L, the buffer of 1 μ L, the XbaI enzyme of 0.5 μ L and the ddH of 6.5 μ L₂O。

In step B1, the cumulative volume of described PCR reaction system is 50 μ L: include the Ex Taq of 0.25 μ L Enzyme, the Ex Taq buffer of 5 μ L, the dNTP of 4 μ L, the template of 2 μ L and 4 μ L primers, remaining Amount is aquesterilisa, and the concentration of the dNTP used is 2.5mM, and the concentration of template is 100ng/ μ L, primer Concentration be 10uM.

In step B1, the PCR amplification program of use is: carrying out 25 circulations, the most each circulation is successively Comprising the following steps: denaturation 10s at a temperature of 98 DEG C, anneal at a temperature of 50 DEG C 30s；72 DEG C of temperature 2min is extended under degree；After completing 25 circulations, i.e. complete PCR amplification program.

In step B2, the cumulative volume of described PCR reaction system is 50 μ L: include the Ex Taq of 0.25 μ L Enzyme, the Ex Taq buffer of 5 μ L, the dNTP of 4 μ L, the template of 2 μ L and 4 μ L primers, remaining Amount is aquesterilisa, and the concentration of the dNTP used is 2.5mM, and the concentration of template is 100ng/ μ L, primer Concentration be 10uM.

In step B2, the PCR amplification program of use is: carrying out 25 circulations, the most each circulation is successively Comprising the following steps: denaturation 10s at a temperature of 98 DEG C, anneal at a temperature of 50 DEG C 30s；72 DEG C of temperature 2min is extended under degree；After completing 25 circulations, i.e. complete PCR amplification program.

After step step C4 completes, also include that C5, virus titer detect: use virus titer detectable Box detection virus titer.

According to embodiments of the invention, when using the detection of virus titer detection kit, the standard of virus titer Curve is as in figure 2 it is shown, the testing result of virus titer is as shown in table 2:

The invention also discloses, the slow virus that described slow virus preparation method prepares is in infecting T cell Application.

Embodiments of the invention 2, disclose example slow virus infection T cell and an embodiment for effect detection. From embodiment it can be seen that the slow virus prepared by described slow virus preparation method, it may not be necessary to add CD28, only before transfection, directly adds a small amount of CD3 in culture medium and believes as t cell activation first Number, reduce program prepared by Car-T cell, reduce cost and pollution risk；By restructuring VSVG, thorn Sharp T cell activates further, increases virus with cells contacting probability to be effectively improved efficiency of infection.

For the technical scheme in the further clear and definite present invention, make following description, do not carry out stressing, It is industry universal term, is not repeating at this.

CD137 (4-1BB) is the cell expression molecule of the mankind and mouse 4-1BB homology, is I type glycoprotein, Belong to Tumor Necrosis Factor Receptors (TNFR) superfamily member, be that another in addition to CD28/B7 is important The costimulatory molecules of mediating T-cell activation, CD137 can activate the JNK in downstream, p38, MAPK and The signal pathways such as NFkB, play Graft Versus Tumor by following approach: strengthen tumour-specific T thin The propagation of born of the same parents and activation；Improve the tumor tropism of tumor specific T cells；Promote tumor antigen Memorability The generation of CD8+T cell；Stop the death of activation inducing T cell.Using CD137 as stimulating T cell The synergistic signal of activation, in conjunction with slow virus packaging and the improvement of T cell activation mode, recombinates in VSVG, Together it is packaged into slow virus with targeting vector, is respectively provided with positive effect with long-term activating T cell aspect in early days. The slow virus carrying 4-1BB-VSVG can be combined with the CD137 of T lymphocytic cell surface, strengthens slow sick Poison contacts with T lymphocyte, is conducive to improving slow virus infection efficiency.

In the present invention, the nucleotide sequence of VSVG is as shown in SEQ ID NO:1；

The structure chart of VSVG is as shown in Figure 3；

The nucleotide sequence of 4-1BB is as shown in SEQ ID NO:2；

The nucleotide sequence of 4-1BB-VSVG is as shown in SEQ ID NO:3；

The aminoacid sequence of 4-1BB-VSVG is as shown in SEQ ID NO:4；

The structure chart of 4-1BB-VSVG is as shown in Figure 4.

Accompanying drawing explanation

Fig. 1 is the schematic diagram of T cell activation process in prior art；

When Fig. 2 is to use the detection of virus titer detection kit in embodiment, the canonical plotting of virus titer；

Fig. 3 is the structure chart of VSVG；

Fig. 4 is the structure chart of 4-1BB-VSVG；

Fig. 5 is the result figure of efficiency of infection in embodiment 2；

Fig. 6 is the result figure of CD69 positive expression rate in embodiment 2.

Detailed description of the invention

Below in conjunction with embodiment, further illustrate present disclosure.Should be appreciated that the enforcement of the present invention Being not limited to the following examples, any pro forma accommodation and/or the change of being made the present invention all will fall Enter scope.

In the present invention, if not refering in particular to, all of part, percentage ratio are unit of weight, all of equipment and Raw materials etc. are all commercially available or the industry is conventional.Method in following embodiment, as without saying especially Bright, it is the conventional method of this area.

Embodiment 1

A, the structure of slow virus carrier plasmid 881-3-SJ19:

In step A1, the cumulative volume of described PCR reaction system is 50 μ L: include the Ex Taq of 0.25 μ L Enzyme, the Ex Taq buffer of 5 μ L, the dNTP of 4 μ L, the template of 2 μ L and 4 μ L primers, remaining Amount is aquesterilisa, and the concentration of the dNTP used is 2.5mM, and the concentration of template is 100ng/ μ L, primer Concentration be 10uM；

The PCR amplification program used is: carrying out 25 circulations, the most each circulation includes following step successively Rapid: denaturation 10s at a temperature of 98 DEG C, anneal at a temperature of 50 DEG C 30s；Extend at a temperature of 72 DEG C 2min；After completing 25 circulations, i.e. complete PCR amplification program.

A5, enzyme action detect: carry out enzyme action step successively and coupled reaction detects described 881-3-SJ19 plasmid Sequence；The enzyme action system used is: the 881-3-SJ19 plasmid of 2 μ L, the buffer of 1 μ L, 0.5 μ L XhoI enzyme and the ddH of 6.5 μ L₂O；The linked system used is: the slow virus carrier of 2 μ L, 1 μ L Buffer, the XbaI enzyme of 0.5 μ L and the ddH of 6.5 μ L₂O

Wherein, the operation of gel purification CD19CAR amplified production comprises the following steps:

4. after gel is completely dissolved, observe the color of sol solutions, if sol solutions color from yellow becomes orange Color or pink colour, add 3M sodium acetate solution (pH5.2) 10 μ l in above-mentioned blob of viscose lysate, uniformly mix Yellow is recovered to solution.When separating the DNA fragmentation less than 400bp, end should be added in this solution Concentration is the isopropanol of 20%.

5. the Spin Column in test kit is placed on Collection Tube.

8. add the BufferWB of 700 μ l, Spin Column be placed on Collection Tube, Room temperature 12,000rpm is centrifuged 1 minute.

9. Spin Column is placed on the centrifuge tube of new 1.5ml, in the central authorities of Spin Column film Place adds 30 μ l sterile purified waters or Elution Buffer (is heated to during 60 DEG C of uses being conducive to improving eluting effect Rate.) room temperature stand 1 minute.Room temperature 12000rpm is centrifuged 1 minute eluted dna.

Wherein, Buffer GM usage amount is shown in Table 1

Table 1:Buffer GM uses scale

Gel strength	Buffer GM usage amount
		1.0%	3 gelinite accumulated amounts
1.0% 1.5%	4 gelinite accumulated amounts
		1.5% 2.0%	5 gelinite accumulated amounts

The transformation of B, VSVG plasmid:

The cumulative volume of described PCR reaction system is 50 μ L: include the Ex Taq enzyme of 0.25 μ L, 5 μ L Ex Taq buffer, the dNTP of 4 μ L, the template of 2 μ L and 4 μ L primers, surplus is sterilizing Water, the concentration of the dNTP used is 2.5mM, and the concentration of template is 100ng/ μ L, and the concentration of primer is 10uM。

C, the preparation of slow virus:

C3, cell transfecting and cultivation: carry the last fortnight recovery 293T cell, train in 10cm culture dish Support, every day observation of cell；Treat that cell is paved with about 2 layers, 1 dish cell dissociation is passaged to 10 dishes, every day Observe, reach about 70% Shi Ke at the bottom of ware to cell density and transfect.Before transfection, with 8ml without serum DMEM culture medium changes liquid；After changing liquid, being added dropwise over the transfection reagent that step C2 obtains, slowly piping and druming is mixed Even above transfection reagent, often dish cell takes this reagent of 1ml, is dropwise homogeneously added into the 293T having changed liquid In cell.Culture dish fore-and-aft direction, left and right directions are rocked the most gently tens of mixings, cell is put into training Support case, stand 2 hours.Often dish cell adds 1ml hyclone, after being dropwise homogeneously added into, will cultivate Ware fore-and-aft direction, left and right directions rock tens of mixings the most gently, and cell is put into incubator.After about 8h, Often the dish cell 10ml DMEM containing 10% hyclone changes liquid, the CO2 concentration of incubator is adjusted to 3%；

C4, collection virus: after step C3 completes, collect supernatant (100ml altogether), and 1200rpm is centrifuged 5min, 0.22um (PES) membrane filtration supernatant, is virus stock solution used.

C5, virus titer detect: detect virus titer by virus titer detection kit, and testing result is about 10⁸/ ml, the standard curve of virus titer is as in figure 2 it is shown, the testing result of virus titer is as shown in table 2:

Table 2: the testing result table of virus titer

Sample name	OD value	Detection titer (IFU/ml)	Actual titer
				Virus stock solution used (dilution 10³Times)	1.777	1141.320	1.141*10⁶
Virus stock solution used (dilution 10⁴Times)	0.311	125.969	1.260*10⁶
				(the dilution 10 of viral concentration liquid⁵Times)	1.689	1080.371	1.080*10⁸
(the dilution 10 of viral concentration liquid⁶Times)	0.293	113.502	1.135*10⁸

Embodiment 2

Slow virus infection T cell and effect detection

1, acquisition about 1 × 10 adopted by machine⁷Individual monokaryon lymphocyte；Ficoll isolates mononuclearcell, adds containing IL-2 (1000U/ml) vivo15 culture medium is cultivated, and adding a small amount of CD3 antibody stimulates (100ug/ml). Before Gan Raning, cell concentration is adjusted to 1 × 10⁶/ ml, takes volume 1ml, cultivates in 6 orifice plates；Come by MOI=5 Calculating virus quantity, before adding viral concentration liquid, add 1ml complete culture solution and suspend, 0.22um filters.Add After entering virus, mix gently, after putting into incubator cultivation 3 days, wash away virus and continue to cultivate.

2, efficiency of infection detection

Collect 5 × 10⁵Cell；Three times are washed with buffer (PBS+2%BSA)；Cell is resuspended in 200 μ L's Buffer, adds the protein L-biotin of 1ug, 4 DEG C of 45min；Wash once with buffer；Cell is resuspended in 200 The buffer of μ L, adds the SA-PE (5ug/ml) of 10 μ L, adds 2 μ LCD3 APC simultaneously, lucifuge, 4℃25min；Wash once with buffer, be suspended into 200 μ L with buffer；Flow cytometer detection PE and APC Fluorescence.After VSVG restructuring, efficiency of infection is improved to 65.18% by 27.41%, and result is as shown in Figure 5.

3, activation efficiency detection

Collect 2 × 10⁵Cell；Three times are washed with buffer (PBS+2%BSA)；Cell is resuspended in 200 μ L Buffer, add the CD69-FITC of 2ug, 4 DEG C of lucifuges hatch 30min；Wash once with buffer, use Buffer is suspended into 200 μ L；Flow cytometer detection fluorescence.Activation efficiency is about 45%, it is seen that improved slow disease Poison can effectively activate T cell, and result is as shown in Figure 6.

It should be noted last that, above example only in order to technical scheme to be described and unrestricted, Although the present invention being described in detail with reference to preferred embodiment, be it should be understood that and be the foregoing is only The detailed description of the invention of the present invention, the protection domain being not intended to limit the present invention, all the present invention's Within spirit and principle, any modification, equivalent substitution and improvement etc. done, should be included in the present invention's Within protection domain.

Claims

1. the slow virus preparation method comprising 4-1BB, it is characterised in that comprise the following steps:

A, the structure of slow virus carrier plasmid 881-3-SJ19:

The transformation of B, VSVG plasmid:

C, the preparation of slow virus:

C2, preparation transfection reagent: 881-3-SJ19 plasmid that step A4 is obtained, NRF plasmid, step 4-1BB-VSVG plasmid that B5 obtains, 1M CaCl₂And distilled water, after mix homogeneously, it is added dropwise over step The BES solution that rapid C1 obtains, after dropping, stands 30min；Obtain transfection reagent；

Slow virus preparation method the most according to claim 1, it is characterised in that in step A1, institute The cumulative volume stating PCR reaction system is 50 μ L: include the Ex Taq enzyme of 0.25 μ L, the Ex Taq of 5 μ L Buffer, the dNTP of 4 μ L, the template of 2 μ L and 4 μ L primers, surplus is aquesterilisa, is used The concentration of dNTP be 2.5mM, the concentration of template is 100ng/ μ L, and the concentration of primer is 10uM.

Slow virus preparation method the most according to claim 1, it is characterised in that in step A1, make PCR amplification program be: carrying out 25 circulations, the most each circulation comprises the following steps successively: Denaturation 10s at a temperature of 98 DEG C, anneal at a temperature of 50 DEG C 30s；2min is extended at a temperature of 72 DEG C；? After completing 25 circulations, i.e. complete PCR amplification program.

Slow virus preparation method the most according to claim 1, it is characterised in that step A4 completes it After, also include that A5, enzyme action detect: carry out enzyme action step successively and coupled reaction detects described 881-3-SJ19 The sequence of plasmid；Use enzyme action system be: the 881-3-SJ19 plasmid of 2 μ L, the buffer of 1 μ L, The XhoI enzyme of 0.5 μ L and the ddH of 6.5 μ L₂O；The linked system used is: the slow virus carrier of 2 μ L, The buffer of 1 μ L, the XbaI enzyme of 0.5 μ L and the ddH of 6.5 μ L₂O。

Slow virus preparation method the most according to claim 1, it is characterised in that in step B1, institute The cumulative volume stating PCR reaction system is 50 μ L: include the Ex Taq enzyme of 0.25 μ L, the Ex Taq of 5 μ L Buffer, the dNTP of 4 μ L, the template of 2 μ L and 4 μ L primers, surplus is aquesterilisa, is used The concentration of dNTP be 2.5mM, the concentration of template is 100ng/ μ L, and the concentration of primer is 10uM.

Slow virus preparation method the most according to claim 1, it is characterised in that in step B1, make PCR amplification program be: carrying out 25 circulations, the most each circulation comprises the following steps successively: Denaturation 10s at a temperature of 98 DEG C, anneal at a temperature of 50 DEG C 30s；2min is extended at a temperature of 72 DEG C；? After completing 25 circulations, i.e. complete PCR amplification program.

Slow virus preparation method the most according to claim 1, it is characterised in that in step B2, institute The cumulative volume stating PCR reaction system is 50 μ L: include the Ex Taq enzyme of 0.25 μ L, the Ex Taq of 5 μ L Buffer, the dNTP of 4 μ L, the template of 2 μ L and 4 μ L primers, surplus is aquesterilisa, is used The concentration of dNTP be 2.5mM, the concentration of template is 100ng/ μ L, and the concentration of primer is 10uM.

Slow virus preparation method the most according to claim 1, it is characterised in that in step B2, make PCR amplification program be: carrying out 25 circulations, the most each circulation comprises the following steps successively: Denaturation 10s at a temperature of 98 DEG C, anneal at a temperature of 50 DEG C 30s；2min is extended at a temperature of 72 DEG C；? After completing 25 circulations, i.e. complete PCR amplification program.

Slow virus preparation method the most according to claim 1, it is characterised in that step step C4 is complete After one-tenth, also include that C5, virus titer detect: detect virus titer by virus titer detection kit.

10. the slow virus that the arbitrary described slow virus preparation method of claim 1-9 prepares is thin at infection T Application in born of the same parents.