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Lentivirus packaging method

A technology of lentivirus packaging and packaging method, which is applied in the field of lentivirus packaging, and can solve the problems of unstable virus yield in batches and low lentivirus titer, etc.

Pending Publication Date: 2022-05-10
GUANGDONG PROCAPZOOM BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In most cases, the current lentivirus is obtained by transiently transfecting the packaging plasmid and the expression plasmid into cells, and the titer of the obtained lentivirus is low, and the amount of virus produced between virus batches is unstable

Method used

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  • Lentivirus packaging method

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Effect test

Embodiment 1

[0030] A packaging method for lentivirus, comprising the following steps:

[0031] (1) Divide about (1~10)×10 6 293T cells were inoculated into T75 culture flasks, added with DMEM complete medium containing 10vol% fetal bovine serum and 1vol% penicillin-streptomycin, cultured overnight, when the cell density reached 50-90%, the culture flasks were discarded. To the supernatant, add 10 mL of DMEM medium containing only 0.1 vol% fetal bovine serum to starve the cells for 1 hour to obtain tool cells;

[0032] (2) Aspirate the Opti-MEM medium according to the amount of 1-10 mL of each virus, and divide it into two equal parts. Add lentiviral packaging plasmids psPAX2, pMD2.G and target expression plasmids to one part, mix well to obtain a plasmid mixture; add polyethylenimine to another part, mix well, and then place at room temperature for 1-10 minutes to obtain a transfection reagent;

[0033] Among them, the mass ratio of psPAX2, pMD2.G, and the target expression plasmid is 1...

Embodiment 2

[0038] A packaging method for lentivirus, comprising the following steps:

[0039] (1) Divide about (1~10)×10 6 293T cells were inoculated into T75 culture flasks, added with DMEM complete medium containing 10vol% fetal bovine serum and 1vol% penicillin-streptomycin, cultured overnight, when the cell density reached 50-90%, the culture flasks were discarded. To the supernatant, add 10 mL of DMEM medium containing only 0.5 vol% fetal bovine serum to starve the cells for 1 hour to obtain tool cells;

[0040] (2) Aspirate the Opti-MEM medium according to the amount of 1-10 mL of each virus, and divide it into two equal parts. Add lentiviral packaging plasmids psPAX2, pMD2.G and target expression plasmids to one part, mix well to obtain a plasmid mixture; add polyethylenimine to another part, mix well, and then place at room temperature for 1-10 minutes to obtain a transfection reagent;

[0041] Among them, the mass ratio of psPAX2, pMD2.G, and the target expression plasmid is 3...

Embodiment 3

[0046] A packaging method for lentivirus, comprising the following steps:

[0047] (1) Divide about (1~10)×10 6 293T cells were inoculated into T75 culture flasks, added with DMEM complete medium containing 10vol% fetal bovine serum and 1vol% penicillin-streptomycin, cultured overnight, when the cell density reached 50-90%, the culture flasks were discarded. To the supernatant, add 10 mL of DMEM medium containing only 1 vol% fetal bovine serum to starve the cells for 1 hour to obtain tool cells;

[0048] (2) Aspirate the Opti-MEM medium according to the amount of 1-10 mL of each virus, and divide it into two equal parts. Add lentiviral packaging plasmids psPAX2, pMD2.G and target expression plasmids to one part, mix well to obtain a plasmid mixture; add polyethylenimine to another part, mix well, and then place at room temperature for 1-10 minutes to obtain a transfection reagent;

[0049] Among them, the mass ratio of psPAX2, pMD2.G, and the target expression plasmid is 5:2...

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Abstract

The invention provides a lentivirus packaging method which comprises the following steps: (1) adding plasmids into a serum-free culture medium to obtain a plasmid mixture; the plasmids comprise lentivirus packaging plasmids and target expression plasmids; the lentivirus packaging plasmids comprise psPAX2 and pMD2. G; (2) adding the transfection medium into a serum-free culture medium to obtain a transfection reagent; and (3) uniformly mixing the plasmid mixture and a transfection reagent, adding the mixture into the tool cells, transfecting and culturing for 2-10 hours, replacing the culture medium with a serum-reduced culture medium, continuously culturing, collecting a supernatant, and concentrating to obtain the lentivirus. In the lentivirus packaging method provided by the invention, the target expression plasmid and the two packaging plasmids are utilized to carry out co-transfection on the tool cells, so that the lentivirus can stably package the tool cells, and the lentivirus packaging efficiency and the lentivirus titer are improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a packaging method of lentivirus. Background technique [0002] Lentivirus is a kind of retrovirus, which can integrate the target gene into the genome of the host and achieve stable and long-lasting expression. It has high safety and has higher titer and more Wide host range, capable of infecting both dividing and non-dividing cells. After the lentiviral genome enters the cell, it is reverse-transcribed into DNA in the cytoplasm to form a pre-DNA integration complex, which is integrated into the genome of the host cell after entering the nucleus, and the integrated DNA is transcribed to generate mRNA, which returns to the cytoplasm, and finally Express target protein or produce small RNA. [0003] Lenti vector is a gene therapy vector developed on the basis of human immunodeficiency virus type 1 (HIV-1). Compared with transient expression vectors, lentiviral vectors have extremel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N7/01
CPCC12N15/86C12N7/00C12N2740/15021C12N2740/15043C12N2740/15052C12N2800/107
Inventor 曾皓宇沈振波蒋碧愉刘康志赵燕玲
Owner GUANGDONG PROCAPZOOM BIOSCIENCES CO LTD
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