Recombinant lentivirus production kit and application thereof
A technology of recombinant lentivirus and kit, applied in the field of genetic engineering, can solve the problems of affecting virus recovery efficiency, virus activity, restrict application and popularization, etc., and achieve the effects of increasing virus yield, reducing production cost, and lifting application restrictions.
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Embodiment 1
[0037] Example 1 Recombinant lentivirus production kit
[0038] Recombinant lentivirus production kit includes:
[0039] Packaging plasmid mixture: the packaging plasmid mixture is a mixture of pMD.2G plasmid and pSPAX2 plasmid, and the mass ratio between them is 1:2.
[0040] Transfection reagent A, the transfection reagent A is 0.5-3 mg / ml polyetherimide (purchased from Polysciences) solution.
[0041] Preparation of the packaging plasmid mixture:
[0042] 1. Strain recovery: add 5 μl of the strains containing the helper plasmid pMD.2G plasmid (pH1) and pSPAX2 plasmid (pH2) to 5 ml of LB medium containing 50 μg / mL ampicillin, and cultivate on a shaker at 37°C and 200 rpm 8h.
[0043] 2. Shaking bacteria: Take 100 μl of the recovered strain and add it to 100 ml LB medium containing 50 μg / mL ampicillin, culture on a shaker at 37° C. at 200 rpm, and carry out plasmid extraction and purification when OD600 = 2.0.
[0044]3. Plasmid extraction: extract the plasmid according t...
Embodiment 2
[0046] Example 2 Recombinant lentivirus production kit
[0047] Recombinant lentivirus production kit includes:
[0048] Packaging plasmid mixture: the packaging plasmid mixture is a mixture of pMD.2G plasmid and pSPAX2 plasmid, and the mass ratio between the two is 1:1. The preparation method of the packaging plasmid mixture is as described in Example 1.
[0049] Transfection reagent A, the transfection reagent A is 0.5-2mg / ml polyetherimide aqueous solution.
[0050] Coating reagent B, the coating reagent B is a solution containing polylysine and fibronectin, wherein the concentration of polylysine is 0.05-1 mg / ml, and the concentration of fibronectin is 0.05-0.25 mg / ml.
[0051] Synergistic reagent C, the synergistic reagent C is a solution containing sodium butyrate and valproic acid, wherein the concentration of sodium butyrate is 0.1-0.4 mg / ml, and the concentration of valproic acid is 0.25-0.5 mg / ml.
[0052] Concentrated reagent D, said concentrated reagent D is a s...
Embodiment 3
[0053] The process of embodiment 3 lentivirus production
[0054] Utilize the kit described in embodiment 2, press figure 1 The production process shown is for the production of lentivirus, and the specific steps include:
[0055] 1. Preparation of carrier plasmids carrying genes of interest
[0056] The carrier plasmid carrying the target gene was prepared in advance. In this embodiment, the pCDH vector plasmid was selected, and EGFP was selected as the target gene for experiments to prepare the pCDH-EF1-EGFP plasmid (carrier plasmid carrying the gene target gene).
[0057] 2. 293T cell preparation:
[0058] Take out a frozen 293T cell (purchased from ATCC) from liquid nitrogen and quickly put it in a 37°C water bath until the ice cube disappears, add it dropwise to a 15ml centrifuge tube containing 5ml preheated medium, centrifuge at 1200rpm for 3min, discard Clear, resuspend the cells in 293T medium (10% FBS + 1mM sodium pyruvate + 2mM glutamine + 1% non-essential amino ...
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