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Lentivirus stable packaging cell line and preparation method thereof

A technology for packaging cell lines and lentiviruses, which is applied in the fields of immunology and molecular biology, and can solve the problems of large residues of impurities, unstable virus production, and low lentivirus titers.

Active Publication Date: 2021-03-02
BEIJING IMMUNOCHINA MEDICAL SCI & TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the titer of the lentivirus produced by the current production process is low, the residue of impurities is large, and the amount of virus produced between batches of viruses is also unstable.

Method used

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  • Lentivirus stable packaging cell line and preparation method thereof
  • Lentivirus stable packaging cell line and preparation method thereof
  • Lentivirus stable packaging cell line and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Construction of embodiment 1pPuro.coTetR plasmid, pVSVG plasmid and pGagPol-RRE-NES-cINT plasmid

[0095] The lentivirus stable packaging cell line constructed in this example consists of three plasmids, pPuro.coTetR (comprising the CoTetR gene), pVSVG (comprising the VSVG gene), and pGagPol-RRE-NES-cINT (comprising the GagPol and Rev genes), which See the plasmid map of figure 1 a, figure 1 b and figure 1 c.

[0096] pPuro-coTetR vector construction:

[0097] The INT fragment (230bp) was obtained by primer TetR-WF-1F / TetR-1R-linker and template pIRESpuro3 (TaKaRa, product number: 631619), and by primer TetR-2F-linker / TetR-2R-linker and template coTetR (its sequence SEQ ID NO: 1) to obtain the coTetR fragment (624bp), through the primer TetR-3F-linker / TetR-WF-3R and the template pcDNA4 (Thermo, catalog number: V102020) to obtain the PA+F1 ori+SV40 promoter fragment (1042bp) . Then, by bridging PCR, the primer TetR-WF-1F / TetR-2R-linker was used to obtain the fragme...

Embodiment 2

[0108] Example 2 Preparation of lentivirus stable packaging cell line

[0109] The three plasmids constructed in Example 1 were linearized and digested with Fsp I (Thermo, catalog number: FD1224), and then dephosphorylated before being used to transfect 293 cells.

[0110] The linearized pPuro.coTetR and pVSVG plasmids were used to co-transfect 293 suspension cells with PEI (polyethyleneimine) (Polysciences, catalog number: 23966). 6 cells, PEI amount: (pPuro.coTetR plasmid amount+pVSVG plasmid amount)=4:1, after using 2 μg / mL Puro (puromycin) (InVivogen, product number: ant-pr-1), 400 μg / mL Zeo ( Bleomycin) (Invitrogen, product number: R25001) antibiotics were subjected to pressurized selection for 2 weeks to obtain drug-resistant positive stable cell pools, ie polyclonal PV cells. Polyclonal PV cells were plated in a 96-well plate (Thermo, catalog number: 167008) by limiting dilution method, with 200 μL of medium per well. Then observe under the microscope, pick out the we...

Embodiment 3

[0113] Example 3 Identification of stable packaging cell lines by semi-quantitative PCR

[0114] The surviving monoclonal cells in the 96-well plate were expanded and cultured, and 48-well plate, 24-well plate, 12-well plate, 6-well plate and shake flask E125 were used respectively. RNA was extracted from PVGR monoclonal cells, and after reverse transcription into cDNA, semi-quantitative PCR was performed to identify whether the target genes CoTetR, VSVG and GagPol were contained. β-actin was used as an internal reference gene. Specific steps are as follows:

[0115] The PVGR monoclonal cells were plated in a 12-well plate the day before, and 3 × 10 5 cells / mL, add 2 μg / mL dox (doxycycline) (Sigma, product number: D9891) on the second day, and harvest the cells on the third day for RNA extraction. with TRIzol TM Reagents (Invitrogen TM , Cat. No.: 15596026) to extract RNA, and the extraction method was operated according to the instructions.

[0116] The extracted RNA wa...

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Abstract

The invention relates to a lentivirus stable packaging cell line and a preparation method thereof, the lentivirus stable packaging cell line comprises a packaging plasmid group, and the packaging plasmid group is composed of a pPuro.coTetR plasmid, a pVSVG plasmid and a pGagPol-RRE-NES-cINT plasmid, wherein the pPuro.coTetR plasmid comprises a CoTetR gene, the pVSVG plasmid comprises a VSVG gene,and the pGagPol-RRE-NES-cINT plasmid comprises a GagPol gene and a Rev gene. The invention further discloses the preparation method of the recombinant plasmid. The lentivirus stable packaging cell line is relatively stable in the aspects of passage, virus production and genetic gene copy number, and the lentivirus produced by using the lentivirus stable packaging cell line is high in titer and lowin impurity content.

Description

technical field [0001] The invention relates to the fields of immunology and molecular biology, mainly the field of cell therapy, in particular to a lentivirus stable packaging cell line and a preparation method thereof. Background technique [0002] Lentivirus is a gene therapy vector, including Human Immunodeficiency Virus (HIV), Equine Infectious Anemia Virus (EIA), Simian Immunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV), etc. Lentiviral vectors, unlike other retroviral vectors, can infect both dividing and non-dividing cells. Lentivirus can infect various types of cells such as cardiomyocytes, tumor cells, neuron cells, stem cells, hepatocytes, endothelial cells, etc. It can accommodate large foreign fragments, achieve stable expression of target genes, and have a small immune response, so as to achieve good gene expression. therapeutic effect and has broad application prospects. [0003] Current lentiviruses are in most cases produced by transiently t...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N7/01C12R1/91
CPCC12N15/86C12N7/00C12N2740/15021C12N2740/15043C12N2740/15052C12N2740/16043C12N2740/16052C12N2830/006C12N15/85
Inventor 齐菲菲鲁薪安何霆邓翠云
Owner BEIJING IMMUNOCHINA MEDICAL SCI & TECH CO LTD
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