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A kind of efficient lentivirus production system and production method thereof

A production method and lentivirus technology, applied in the directions of viruses/phages, biochemical equipment and methods, viruses, etc., can solve the problems of great difference, different usage ratios and dosages, and the decline of packaging virions, so as to reduce the impact. Effect

Active Publication Date: 2019-07-19
盈凯赛威(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] These four plasmids have different gene segments, especially the packaging plasmids need to be used together to transfect into the target cells, but the usage ratio and dosage are different, and there is a big difference, and there is no conclusion
Commercially packaged plasmids are mostly sold in the form of mixtures, and there is no clear ratio
Moreover, studies have shown that in practical applications, with the extension of the insert fragment, the virus titer decreases in a semi-logarithmic form, and the longer the target plasmid is, the lower the ability to package active virions

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, virus production:

[0031] Spread Lenti-X cells (Liuhetong) into 15cm cell culture dishes in batches 20-24 hours in advance, 9.0X 10 per dish 6 For each cell, the medium used was 25 ml DMEM medium (Gibco, C11995500BT) containing 10% FBS. Place cells at 37°C, 5% CO 2 and 100% humidity conditions.

[0032] Transfection was performed when the cell volume covered 70-80% of the dish. Dissolve PEI at a final concentration of 36ug / ml and DNA at 12ug / ml into DMEM, mix well and incubate at room temperature for 15min. The ratio of the plasmids used is PLP1:PLP2:VSVG:pCDH-EF1-MCS-T2A-copGFP=9:6: 9:8. pCDH-EF1-MCS-T2A-copGFP was purchased from SBI. Pipette 2.5ml of DNA / PEI mixture and slowly add to the edge of the petri dish, shake gently to mix.

[0033] After 6 hours, carefully aspirate the medium and add 25 ml of DMEM containing 2% FBS and 0.12% sodium butyrate. Virus-containing supernatants were collected after 64 hours and awaited concentration.

Embodiment 2

[0034] Embodiment 2, virus concentration:

[0035] Concentration uses a KrosFLO TFF system concentrator from Spectrumlabs. After assembling the system according to the diagram, monitor the pipeline for liquid leakage and check for damage to the hollow fiber. Fill the entire liquid flow system with liquid first, then drain the liquid, turn off the peristaltic pump, reduce the pump speed, and close the backflow and measurement flow with clamps. Turn on the pump to make the column pressure reach 15PSI, close the inlet pressure flow with a clip, turn off the peristaltic pump, and open the side flow clip. If the column pressure drops less than 0.1PSI within one minute, it means that the hollow fiber column is not damaged.

[0036] Wash the column with 1L of water, with a back pressure of 4PSI. After completion, equilibrate the column with 500ml of PBS, and start to concentrate the sample. Be careful not to generate air bubbles during the whole process.

[0037] The collected supe...

Embodiment 3

[0038] Embodiment 3, virus titer detection

[0039] 1. Virus liquid dilution: Centrifuge the concentrated virus at 138,000 g for 2 min, and take the supernatant. Take 50 μl virus supernatant and add to 450 μl DMEM medium, mix well, mark as 10 -1 . from 10-1 The mixed solution was serially diluted with DMEM for 6 dilutions, marked as 5X10 -2 , 2.5X10 -2 , 1.25X10 -2 , 6.25X10 -3 , 3.125X10 -3 , 1.5625X10 -3 .

[0040] 2. Preparation of 293T cells (Shanghai Cell Bank): 293T cells were digested into single cells with Trypsin, washed 2 times and then repopulated with complete medium (DMEM, Gibco, C11995500BT; FBS, Gibco, 10270-106; Penstrept, Gibco, 15140122) Take an appropriate amount of cell suspension and count on a cell counting plate. After counting, dilute the cell suspension to 4X10 with complete medium 5 Cells / ml, according to the volume, add 0.6 μl Polybrene (Shanghai Yisheng Biology, 40804ES76) to each ml.

[0041] 3. Virus infection: Add 500 μl of the above c...

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PUM

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Abstract

The invention provides an efficient lentivirus production system and a production method thereof. The lentivirus production system is a four-plasmid expression system consisting of a vector plasmid carrying an exogenous gene and three packaging plasmids pLP1, pLP2-rev and pLP-VSVG, wherein the packaging plasmid pLP1 expresses a basic structural gene gag / pol of a lentivirus; the packaging plasmid pLP2 expresses a regulatory gene rev of the lentivirus; the packaging plasmid pLP-VSVG expresses an envelope protein gene VSVG of the lentivirus; the ratio of the four plasmids pLP1 to pLP2-rev to pLP-VSVG to the vector plasmid is 9 to 6 to 9 to 8. By use of the lentivirus production system and the production method thereof provided by the invention, high-titer virus with order magnitude up to 109pfu / ml can be obtained.

Description

technical field [0001] The invention belongs to the field of virus preparation, and in particular relates to an efficient lentivirus production system and a production method thereof. Background technique [0002] Lentiviruses belong to the Retroviridae family. Lentivirus vectors are gene therapy vectors developed based on HIV-1 (Human Immunodeficiency Virus Type I), which can effectively integrate foreign DNA or RNA into dividing cells and non-dividing cells. On the host chromosome, and form a stable and long-lasting expression. Compared with general retroviral vectors, lentiviral vectors have the following advantages: they can infect dividing cells and non-dividing cells, including difficult-to-transfect primary cells, stem cells, etc.; The expression time is long; the safety is high, and it is not easy to induce host immune response. Therefore, at the level of gene therapy, lentiviral vectors are being used more and more widely. [0003] The core of HIV-1 lentivirus is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N5/10
CPCC07K14/005C12N5/0602C12N7/00C12N2510/02C12N2740/15022C12N2740/15051
Inventor 高山
Owner 盈凯赛威(北京)生物科技有限公司