CN108893492B - Recombinant lentivirus production kit and application thereof - Google Patents
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Abstract
The invention discloses a recombinant lentivirus production kit and application thereof. The kit comprises a packaging plasmid mixture, a transfection reagent, a synergistic reagent and a coating reagent; the transfection reagent is 0.5-5mg/ml polyetherimide solution; the synergistic reagent is a solution containing sodium butyrate and valproic acid, wherein the concentration of the sodium butyrate is 0.01-1mg/ml, and the concentration of the valproic acid is 0.01-1 mg/ml; the coating reagent is a solution containing polylysine and fibronectin, wherein the concentration of polylysine is 0.01-2mg/ml, and the concentration of fibronectin is 0.01-0.5 mg/ml. The kit provided by the invention is simple to operate, efficient to package and low in cost when in use. The kit is used for producing the recombinant lentivirus, is economic, efficient and convenient, and ensures that the titer of the recombinant lentivirus produced by single-vessel adherent cells reaches 108TU/ml above.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant lentivirus production kit and application thereof.
Background
Lentivirus (Lentivirus) vectors are gene therapy vectors developed based on HIV-1 (human immunodeficiency virus type I), and are distinguished from conventional retroviral vectors, which have the ability to infect both dividing and non-dividing cells and are capable of efficiently transferring a gene of interest (or RNAi) into animal and human primary cells or cell lines.
Lentiviral expression vectors contain the genetic information required for packaging, transfection, and stable integration. The lentiviral packaging plasmid provides all of the helper proteins necessary for transcription and packaging into the recombinant pseudoviral vector. In order to generate high-titer virus particles, cells are co-transfected by an expression vector and a packaging plasmid at the same time, the virus is packaged in the cells, the packaged pseudovirus particles are secreted to a culture medium outside the cells, after centrifugation, a supernatant is obtained and can be directly used for infection of the host cells, and a target gene enters the host cells and is integrated into a genome through reverse transcription, so that effector molecules are expressed at a high level.
In the process of preparing recombinant lentivirus, the titer of the virus is usually used to measure the success of preparing recombinant lentivirus and the quality of the recombinant lentivirus. However, the virus titer after purification in a single culture dish which can be taken by the lentivirus produced adherently or produced in small scale is more than 106-107TU/mL, but this titer of recombinant lentivirus greatly limits the clinical or related applications of recombinant lentivirus. If the virus titer of a single culture dish reaches 108TU/mL requires the use of high quality transfection reagents (such as Lipofectamine series) or transient transfection of 293 or 293T cells in a cell factory, or the use of hollow fiber tube reactors, suspension culture and improved stable cell lines, but this approach is too costly to produce. The conventional methods for concentrating and purifying the lentivirus particles mainly comprise ultracentrifugation, ultrafiltration concentration and addition of a sedimentation reagent. Ultracentrifugation requires special low-temperature high-speed centrifugation equipment, is expensive and can treat a very small amount of virus at a time, and cannot be efficiently used for large-scale virus production. Although a special centrifuge is not needed in ultrafiltration concentration, the virus amount in each centrifugation concentration is limited, and an ultrafiltration column for concentration needs to be replaced in time, so that the ultrafiltration concentration method is not suitable for large-scale virus generation. The settling agent is convenient to use, but the existing settling system can influence the virus recovery efficiency and the activity of the recovered virus sometimes. In summary, the above contents directly or indirectly limit the application and popularization of the related technology using lentivirus as a gene editing vector.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention aims to provide a kit for producing lentivirus, which is simple to operate, efficient to package and low in cost.
Therefore, the technical scheme of the invention is as follows:
a recombinant lentivirus production kit comprises a packaging plasmid mixture, a transfection reagent, a synergistic reagent and a coating reagent;
the transfection reagent is 0.5-5mg/ml polyetherimide solution;
the synergistic reagent is a solution containing sodium butyrate and valproic acid, wherein the concentration of the sodium butyrate is 0.01-1mg/ml, and the concentration of the valproic acid is 0.01-1 mg/ml;
the coating reagent is a solution containing polylysine and fibronectin, wherein the concentration of polylysine is 0.01-2mg/ml, and the concentration of fibronectin is 0.01-0.5 mg/ml.
In the kit, preferably, the transfection reagent is 0.5-3mg/ml polyetherimide solution; most preferably, the transfection reagent is a 0.5-2mg/ml polyetherimide solution.
In the kit, preferably, the concentration of the sodium butyrate in the synergistic reagent is 0.1-0.5mg/ml, and the concentration of the valproic acid is 0.2-0.5 mg/ml; most preferably, the concentration of sodium butyrate in the synergistic agent is 0.1-0.4mg/ml, and the concentration of valproic acid is 0.25-0.5 mg/ml.
In the kit, preferably, the concentration of polylysine in the coating reagent is 0.05-1mg/ml, and the concentration of fibronectin is 0.05-0.25 mg/ml;
the kit also comprises a concentration reagent, wherein the concentration reagent is a solution containing 2mol/L sodium chloride and 0.3-0.6g/mL polyethylene glycol, the mass ratio of PEG6000 to PEG8000 in the polyethylene glycol is 1:1-2, and preferably, the mass ratio of PEG6000 to PEG8000 in the polyethylene glycol is 1: 1.
In the kit, the packaging plasmid mixture is a mixture of a pMD.2G plasmid and a pSPAX2 plasmid, and the mass ratio of the pMD.2G plasmid to the pSPAX2 plasmid is 1: 1-4.
The invention also aims to provide a method for producing recombinant lentivirus, which comprises the following steps:
treating the culture dish with the coating reagent, and culturing the production cells by using the treated culture dish;
preparing a packaging plasmid mixture, co-transfecting the packaging plasmid mixture and a target gene vector plasmid to produce cells under the action of a transfection reagent, and then culturing; the transfection reagent is 0.5-5mg/ml polyetherimide solution;
during the culture of the production cells, the synergist is added when the fresh medium is replaced.
In the above method, the coating agent is a solution containing polylysine and fibronectin, wherein the concentration of polylysine is 0.05-1mg/ml, and the concentration of fibronectin is 0.05-0.25 mg/ml; the synergistic reagent is a solution containing sodium butyrate and valproic acid, wherein the concentration of the sodium butyrate is 0.1-0.5mg/ml, and the concentration of the valproic acid is 0.2-0.5 mg/ml.
In the above method, the transfected production cells are cultured, and after 6-8h of culture, the fresh medium is replaced.
In the above method, the amount of the synergist is 1/(3000-4000) of the volume of the fresh culture medium.
The method further comprises the following steps: after the culture is finished, collecting the supernatant of the cultured production cells, centrifuging, collecting the centrifuged supernatant, mixing the centrifuged supernatant with a concentrated reagent, centrifuging again, and removing the supernatant to obtain the recombinant lentivirus; the concentrated reagent is a solution containing 2mol/L sodium chloride and 0.3-0.6g/mL polyethylene glycol, wherein the mass ratio of PEG6000 to PEG8000 in the polyethylene glycol is 1: 1-2.
In the above method, the packaging plasmid mixture is a mixture of plasmid pmd.2g and plasmid PSPAX2, and the mass ratio between the two is 1: 1-4, for example, 1:1, 1:2, 1:3, 1:4 can be selected.
In the above method, the target gene vector plasmid is a lentiviral vector plasmid, preferably a pCDH vector, an Fuw vector and a pLVX vector;
in the above method, the mass ratio of the target gene vector plasmid to the packaging plasmid mixture is: 1:1-1:2.
In the above method, the producer cell is a 293 line cell, preferably 293T, HEK293, 293F, 293FT, 293E and 293TN, and most preferably 293T.
The invention has the beneficial effects that:
the recombinant lentivirus production kit provided by the invention is simple to operate, high in production efficiency and low in cost when in use. The kit is used for producing the recombinant lentivirus, is economic, efficient and convenient, and ensures that the titer of the recombinant lentivirus produced by single-vessel adherent cells reaches 108TU/ml or more, thereby increasing the virus yield on the basis of reducing the production cost of the recombinant lentivirus, fundamentally removing the application limit of the recombinant lentivirus, and being generally applicable to various lentivirus production systems.
Drawings
FIG. 1 is a flow chart of recombinant lentivirus production.
FIG. 2 fluorescence plot of transfection effect 24 hours after transfection of 293T cells with transfection reagent A.
FIG. 3 is a graph showing the expression of GFP after infecting MSC and Fibroplast cells with pCDH-EF1-EGFP lentivirus under a light and fluorescence microscope.
FIG. 4 fluorescence contrast of transfection effect 24 hours after transfection of 293T cells with different transfection reagents under light and fluorescence.
FIG. 5 is a bar graph comparing viral titers collected at various time points after transfection of 293T cells with different transfection reagents.
FIG. 6 is a bar graph comparing titer of purified virus concentrated by different concentration modes.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below through the embodiments of the present invention and the accompanying drawings, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 recombinant lentivirus production kit
The recombinant lentivirus production kit comprises:
packaging the plasmid mixture: the packaging plasmid mixture is a mixture of a pMD.2G plasmid and a pSPAX2 plasmid, and the mass ratio of the pMD.2G plasmid to the pSPAX2 plasmid is 1: 2.
transfection reagent A, which is a 0.5-3mg/ml polyetherimide (available from Polysciences) solution.
Preparation method of packaging plasmid mixture:
1. recovering strains: mu.l of strain containing the helper plasmids pMD.2G plasmid (pH1) and pSPAX2 plasmid (pH2) was added to 5mL of LB medium containing 50. mu.g/mL ampicillin, respectively, and cultured on a shaker at 37 ℃ and 200rpm for 8 hours.
2. Shaking the bacteria: the recovered strain (100. mu.l) was added to 100mL LB medium containing 50. mu.g/mL ampicillin, and the mixture was cultured on a shaker at 37 ℃ and 200rpm, and plasmid extraction and purification were carried out at OD600 of 2.0.
3. And (3) plasmid extraction: extracting plasmids according to a plasmid extraction method commonly used in the market, and detecting the concentration and the purity of the obtained plasmid DNA by using an ultraviolet spectrophotometer. The purified endotoxin-free DNA plasmid with OD260/OD280 of 1.8-2.0, OD260/230 of 2.1-2.2, supercoiled structure of over 90% and concentration of over 1mg/ml can be directly applied.
4. Preparation of packaging plasmid mixture: and (3) proportioning the pH1 and the pH2 plasmids in the packaging plasmid mixture according to the mass ratio of 1:2, and dissolving with deionized water to obtain a packaging plasmid mixture (helper plasmid premix liquid P).
Example 2 recombinant lentivirus production kit
The recombinant lentivirus production kit comprises:
packaging the plasmid mixture: the packaging plasmid mixture is a mixture of a pMD.2G plasmid and a pSPAX2 plasmid, and the mass ratio of the pMD.2G plasmid to the pSPAX2 plasmid is 1: the preparation of the packaged plasmid mixture is described in example 1.
Transfection reagent A, wherein the transfection reagent A is 0.5-2mg/ml polyetherimide aqueous solution.
And the coating reagent B is a solution containing polylysine and fibronectin, wherein the concentration of polylysine is 0.05-1mg/ml, and the concentration of fibronectin is 0.05-0.25 mg/ml.
And the synergistic reagent C is a solution containing sodium butyrate and valproic acid, wherein the concentration of the sodium butyrate is 0.1-0.4mg/ml, and the concentration of the valproic acid is 0.25-0.5 mg/ml.
And a concentrated reagent D which is a solution of 2mol/L sodium chloride and 0.5g/mL polyethylene glycol, wherein the mass ratio of PEG6000 to PEG8000 in the polyethylene glycol is 1: 1.
Example 3 Process for lentivirus production
The kit of example 2 is used for producing lentivirus according to the production flow shown in figure 1, and the specific steps comprise:
1. preparation of vector plasmid carrying Gene target Gene
In this example, a pCDH vector plasmid was selected and EGFP was selected as a target gene, and an experiment was performed to prepare a pCDH-EF1-EGFP plasmid (a vector plasmid carrying a target gene).
2. Preparation of 293T cells:
1 frozen 293T cell (purchased from ATCC) was rapidly placed in a 37 ℃ water bath from liquid nitrogen until ice disappeared, added dropwise to a 15ml centrifuge tube containing 5ml of a pre-warmed medium, centrifuged at 1200rpm for 3min, the supernatant discarded, the cells were re-suspended with 293T medium (10% FBS +1mM sodium pyruvate +2mM glutamine + 1% non-essential amino acid + DMEM) and inoculated into a 150mM petri dish, 37 ℃ with 5% CO2And (5) culturing at saturated humidity.
Before cell passage, treating a cell culture dish with a coating reagent B, adding a proper amount of the coating reagent B, placing the culture dish at 37 ℃ for incubation for 5min, recovering the coating reagent B, and placing the culture dish in a 37 ℃ incubator for drying for later use. Before cell passage, the dried coated dishes were washed 3 times with PBS.
In the culture process, when the confluency of cells reaches above 90%, subculturing, discarding old culture medium, adding 5ml sterilized PBS solution, shaking gently, washing cells, discarding PBS solution, adding 2ml 0.25%Digesting with Trypsin-EDTA digestive juice for 1-2min until the cells are completely digested. The digestion was stopped by adding serum-containing medium, the cell suspension was centrifuged at 1200rpm for 3min, and the centrifuged cells were resuspended in medium. Cells were seeded at 1.2X 10 per coated 150mm dish7Cells were used for packaging lentiviruses at 37 ℃ with 5% CO2Saturated humidity culture, 20ml medium/dish.
3. Transfection of 293T cells:
changing a 293T cell culture medium into 18ml of a DMEM culture medium 2 hours before transfection, adding 1ml of a preheated DMEM culture medium into an A sterilized centrifuge tube, adding an auxiliary plasmid premix P (packaging plasmid mixture) and pCDH-EF1-EGFP (vector plasmid carrying gene target genes) according to the mass ratio of 1:1, blowing, beating and mixing uniformly. 1ml of pre-heated DMEM medium was added to the B sterile centrifuge tube, followed by 162. mu.l of transfection reagent A (0.5-2mg/ml of polyetherimide) and mixed well. Tubes A and B were incubated at room temperature for 5 min. The liquid in tube B was added dropwise to tube A, mixed well and incubated at room temperature for 10min to form DNA-transfection reagent complexes.
Transferring the DNA-transfection reagent complex to 293T cells with a pre-changed solution, mixing, and performing 5% CO at 37 deg.C2And (5) culturing at saturated humidity. After 6-8h of incubation, the medium containing the transfection mixture was aspirated off, and 20ml of pre-warmed DMEM medium containing 5% FBS and 5ul of booster reagent C (0.2-0.8mg/ml sodium butyrate and 5ul of booster reagent C) were added to each dish of cells
Mixed solution of 0.5-1mg/ml valproic acid), 5% CO at 37 deg.C2And (5) culturing at saturated humidity. After the liquid change, the supernatant liquid is collected for 24h and 48h respectively and temporarily stored at 4 ℃, 20ml of fresh culture medium is changed, and 5ul of the synergistic agent C is supplemented.
Cells collected 24h after transfection were observed under a fluorescence microscope, and the results obtained are shown in FIG. 2. As shown in FIG. 2, after transfection of helper plasmid and pCDH-EF1-EGFP (expressing green fluorescent protein EGFP) into 293T cells by transfection reagent A for 24h, over 99% of 293T cells were positive.
4. Recombinant lentivirus collection and concentration
Centrifuging the collected liquid at 4 ℃ and 3500rpm for 15min, discarding the precipitate, mixing the supernatant with the concentrated reagent D, standing at 4 ℃ for 24h, centrifuging at 4 ℃ and 3000rpm for 30min, discarding the supernatant, resuspending the precipitate into 500 μ l DMEM medium, and performing virus titer determination.
5. Recombinant lentivirus titer assay
1 day before titre assay, 6-well plates were seeded at 1 x 10 per well6Adding 2ml of 293T medium without antibiotics at 37 ℃ and 5% CO into the 293T cells2The culture was carried out overnight under saturated humidity. The next day, the concentrated virus was added, the gradient was set to 0ul (control), 0.1ul, 0.2ul, 0.3ul, 0.4ul and 0.5ul, respectively, and the solution was changed 24 hours after infection. After 72h, cells in a 6-well plate are digested (the method is the same as the cell passage), the GFP cell positive rate is determined by flow analysis, and the group titer test with the positive rate within 20 percent is effective.
The titer (transmission units, TU) was determined using the formula:
positive cell rate × number of seeded cells/0.001 ml-titer (TU/ml).
As can be seen from Table 1, the titers of 3 batches of single-dish lentiviruses produced by using the lentivirus kit are all 108TU/ml above.
TABLE 1 flow cytometry for the titer of three batches of lentiviruses produced by the lentivirus kit
6. Recombinant lentivirus activity assay
The activity of the obtained lentivirus is verified by infecting umbilical mesenchymal stem cells and fibroblasts with the collected recombinant lentivirus particles. Counting and passage are carried out on the cells one day before virus activity verification, and the confluence degree of the umbilical cord mesenchymal stem cells and the fibroblasts cannot exceed 30% on the next day of passage. Umbilical cord mesenchymal cells were infected at a multiplicity of infection (MOI) of 40 and fibroblasts were infected at a multiplicity of infection of 5. The cells were incubated for another 48 hours after 24 hours of infection, and the expression of green fluorescent protein was observed under a fluoroscope, as shown in FIG. 3.
According to the figure 3, when the umbilical cord mesenchymal stem cells and the fibroblasts are infected by the lentiviruses produced by the kit, the two cells can reach the infection efficiency of more than 90%, and the growth state of the cells is not influenced.
Example 4 comparison of the efficiency of different transfection reagents for packaging viruses
The Transfection effect and virus yield of the commonly used high efficiency Transfection Reagent Lipofectamine 3000Transfection Reagent (from Invitrogen, 1.5ml package price is about 6500 yuan), Lipofectamine LTX Reagent with PLUS Reagent (from Invitrogen, 1.0ml package price is about 6500 yuan) and Transfection Reagent A in example 2 (2.0g package price is about 3000 yuan)) were compared.
The 293T cell preparation method, the recombinant lentivirus collection and concentration method and the recombinant lentivirus titer determination method are the same as those in example 3.
Transfection of 293T cells: three different methods were used to transfect 293T cells, as follows:
(1) lipofectamine 3000Transfection Reagent Transfection method:
changing 293T cell culture medium into 18ml DMEM culture medium 2 hours before transfection, adding 1ml preheated DMEM culture medium into an A sterilized centrifuge tube, adding an auxiliary plasmid premix P and pCDH-EF1-EGFP (vector plasmid carrying gene target gene) according to the mass ratio of 1:1, adding a P3000 reagent with the volume 2 times of the total plasmid amount according to the instruction, and uniformly mixing. Add 1ml of pre-heated DMEM medium to the sterile centrifuge tube B, add Lipofectamine 3000 reagent in a volume 1.5 times the total plasmid volume according to the instructions, mix well. Tubes A and B were incubated at room temperature for 5 min. The liquid in tube A was added dropwise to tube B, mixed well and incubated at room temperature for 5min to form DNA-liposome complexes.
(2) Lipofectamine LTX Reagent with PLUS Reagent transfection method:
changing 293T cell culture medium into 18ml DMEM culture medium 2 hours before transfection, adding 1ml preheated DMEM culture medium into the A sterilized centrifuge tube, adding auxiliary plasmid premix P and pCDH-EF1-EGFP (vector plasmid carrying gene target gene) according to the mass ratio of 1:1, adding Plus reagent with the volume 1 time of the total plasmid amount according to the instruction, and mixing uniformly. Add 1ml of pre-heated DMEM medium to the B sterile centrifuge tube, add 1 volume of Lipofectamine LTX reagent to the total plasmid according to the instructions, mix well. Tubes A and B were incubated at room temperature for 5 min. The liquid in tube A was added dropwise to tube B, mixed well and incubated at room temperature for 5min to form DNA-liposome complexes.
(3) Transfection reagent A the transfection method was the same as in example 3.
After the DNA-transfection reagent complexes described in (1), (2) and (3) were formed, the DNA-transfection reagent complexes were transferred to 293T cells previously plated with a medium and plated with reagent B, mixed, incubated at 37 ℃ with 5% CO2And (5) culturing at saturated humidity. After 6-8h of incubation, the medium containing the transfection mixture was aspirated, 20ml of pre-warmed DMEM medium containing 5% FBS and 5ul of booster reagent C were added to each dish of cells, 5% CO at 37 ℃2And (5) culturing at saturated humidity. Cells were harvested 24h after transfection and observed under a fluorescent microscope for green fluorescent protein expression to verify the transfection efficiency of the transfection reagent by comparison (as shown in FIG. 4). Supernatants (multiple time points in one group and multiple times of collection) were collected at 24h, 48h, 72h, 24+48+72h, 48+72h and 24+72h respectively and stored temporarily at 4 ℃, 20ml of fresh culture medium and 5ul of synergistic agent C were added to the group in which the supernatants were collected and fluid changes were required, and the collected viruses were concentrated in the same manner (specifically 4 in example 3) to compare and verify the virus production efficiency at different time points under different transfection methods (as shown in FIG. 4).
According to the figure 4, after the transfection auxiliary plasmid and pCDH-EF1-EGFP (expressing green fluorescent protein EGFP) enter 293T cells for 24h, over 99% of the 293T cells are positive, and the transfection efficiency is not lower than that of a high-price liposome transfection reagent, namely the transfection reagent A has higher transfection capacity.
As can be seen from FIG. 5, in the system of the kit (under the action of the coating reagent B and the synergistic reagent C), the viruses collected at different time points are concentrated and purified, and the titer of the purified viruses is verified, so that the virus production efficiency under the transfection condition of the transfection reagent A at multiple time points is superior to that of other two groups of expensive reagents.
Example 5 comparison of viral efficiencies obtained by different concentration methods
The main comparison between the concentration and purification effects of protein ultrafiltration column (purchased from Millipore) and reagent D in the kit on lentivirus particles in the production process of lentivirus in the laboratory.
The 293T cell preparation method, the method for transfecting the 293T cell, the recombinant lentivirus collection method and the recombinant lentivirus titer determination method are the same as those in example 3.
Concentration of the recombinant lentivirus: equal amounts of virus were collected and the resulting virus suspensions were concentrated by two different methods, respectively, as follows:
(1) the protein ultrafiltration column concentration method comprises the following steps:
15ml of sterile protein ultrafiltration column (Millipore, 30KD) was added with 10ml of sterile PBS and centrifuged through the equilibrated column filter for 10min at 4000rpm at 4 ℃ to ensure that no liquid was present in the upper filter. Sucking 15ml of the collected virus particle suspension, centrifuging at 4 ℃ and 4500rpm for 30min to ensure that the residual liquid in the concentration column is about 500 mu l, and discarding the waste liquid in the lower layer centrifuge tube. The concentration and centrifugation process is repeated until all the collected virus suspension is centrifugally concentrated, and finally the liquid left in the concentration column is adjusted to 500 mul.
(2) Concentration of reagent D the procedure was the same as in example 3.
Finally, the virus collected in (1) and (2) was subjected to titer determination to comparatively verify the virus concentration and purification efficiency of different concentration methods (FIG. 6). As can be seen from fig. 6, in the system of the kit (including a plurality of auxiliary reagents), the titer of the viruses obtained by different concentration and purification methods is verified, and the titer of the viruses obtained by two concentration methods with different initial virus amounts and final concentration and purification methods has no characteristic difference, but the concentration reagent D in the kit has the characteristics of short time, simple operation, economy and practicality when a large amount of viruses are concentrated.
The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (5)
1. A recombinant lentivirus production kit comprises a packaging plasmid mixture and a transfection reagent, and is characterized by also comprising a synergistic reagent and a coating reagent;
the transfection reagent is 0.5-5mg/ml polyetherimide solution;
the synergistic reagent is a solution of sodium butyrate and valproic acid, wherein the concentration of the sodium butyrate is 0.01-1mg/ml, and the concentration of the valproic acid is 0.01-1 mg/ml;
the coating reagent is a solution of polylysine and fibronectin, wherein the concentration of polylysine is 0.01-2mg/ml, and the concentration of fibronectin is 0.01-0.5mg/ml;
the reagent also comprises a concentrated reagent, wherein the concentrated reagent is a solution of 2mol/L sodium chloride and 0.3-0.6g/mL polyethylene glycol, and the mass ratio of PEG6000 to PEG8000 in the polyethylene glycol is 1: 1-2;
the packaging plasmid mixture is a mixture of a pMD.2G plasmid and a pSPAX2 plasmid, and the mass ratio of the pMD.2G plasmid to the pSPAX2 plasmid is 1: 1-4.
2. The kit of claim 1, wherein the transfection reagent is a 0.5-3mg/ml polyetherimide solution; the concentration of sodium butyrate in the synergistic reagent is 0.1-0.5mg/ml, and the concentration of valproic acid is 0.2-0.5 mg/ml; the concentration of polylysine in the coating reagent is 0.05-1mg/ml, and the concentration of fibronectin is 0.05-0.25 mg/ml.
3. A method of recombinant lentivirus production comprising the steps of:
treating the culture dish with the coating reagent, and culturing the production cells by using the treated culture dish;
preparing a packaging plasmid mixture, co-transfecting the packaging plasmid mixture and a target gene vector plasmid to produce cells under the action of a transfection reagent, and then culturing; the transfection reagent is 0.5-5mg/ml polyetherimide solution;
during the culture process of the production cells, when a fresh culture medium is replaced, adding a synergistic reagent, after the culture is finished, collecting the supernatant of the cultured production cells, centrifuging, collecting the centrifuged supernatant, mixing the centrifuged supernatant with a concentrated reagent, centrifuging again, and removing the supernatant to obtain recombinant lentiviruses; the concentrated reagent is a solution of 2mol/L sodium chloride and 0.3-0.6g/mL polyethylene glycol, wherein the mass ratio of PEG6000 to PEG8000 in the polyethylene glycol is 1: 1-2;
the coating reagent is a solution of polylysine and fibronectin, wherein the concentration of polylysine is 0.05-1mg/ml, and the concentration of fibronectin is 0.05-0.25 mg/ml; the synergistic reagent is a solution of sodium butyrate and valproic acid, wherein the concentration of the sodium butyrate is 0.1-0.5mg/ml, and the concentration of the valproic acid is 0.2-0.5 mg/ml;
the packaging plasmid mixture is a mixture of a plasmid PMD.2G and a plasmid PSPAX2, and the mass ratio of the plasmid PMD.2G to the plasmid PSPAX2 is 1: 1-4;
the target gene vector plasmid is a lentiviral vector plasmid, and the lentiviral vector plasmid is a pCDH vector;
the production cell is 293 line cell.
4. The method according to claim 3, wherein the mass ratio of the target gene vector plasmid to the packaging plasmid mixture is: 1:1-1:2.
5. The method of claim 3, wherein the 293 line cells are 293T, HEK293, 293F, 293FT, 293E and 293 TN.
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| Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures;Sven Ansorge et al.;《THE JOURNAL OF GENE MEDICINE》;20090620;第869-870页 * |
| Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures;Laura Cervera1 et al.;《Appl Microbiol Biotechnol》;20151231;第9935-9949页 * |
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