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Lentivirus packaging vector and packaging method

A lentivirus packaging and carrier technology, applied in the direction of viruses/phages, vectors, viruses, etc., can solve the problems of missing exogenous gene sequences, inability to obtain lentivirus particles, interference with lentivirus packaging process, etc., to achieve efficient packaging effect

Pending Publication Date: 2021-01-08
OBIO TECH SHANGHAI CORP LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, research on lentiviral vectors is focused on infectivity, specificity, packaging capacity, etc., but little attention has been paid to the impact of the packaged gene of interest (GOI) on the virus. In practice, a considerable part of the target gene Since self-expression will interfere with the packaging process of lentivirus, it is impossible to obtain effective lentiviral particles
On the other hand, in the lentiviral vector, because the virus packaging process will go through the transcription process, and foreign genes such as circRNA containing important splicing sites will have a large proportion of splicing during the packaging process, so that the final virus Deletion of exogenous gene sequences in granules
There is currently no effective solution for this situation

Method used

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  • Lentivirus packaging vector and packaging method
  • Lentivirus packaging vector and packaging method
  • Lentivirus packaging vector and packaging method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Packaging and titer detection of lentivirus

[0082] 1. Lentiviral packaging

[0083] A method for virus packaging using a lentiviral vector, specifically comprising:

[0084] 1. The day before transfection, inoculate 293T cells into a culture dish; take out the cell culture dish one hour before transfection, discard the original cell culture medium, add Opti-MEM medium, and put the cells back into the incubator; prepare the transfection The complex of staining reagent and plasmid comprises the following steps:

[0085]2. Dissolve the viral vector plasmids to be transfected (skeleton plasmid pCAG-gagpol, envelope protein plasmid pHCMV-VSVG and shuttle plasmid) in Opti-MEM medium, mix gently, and obtain plasmid dilution after standing; The backbone plasmid is pCAG-gagpol, pCAG-gagpol-CMV-CymR or pCAG-gagpol-CMV-TrpR; the envelope protein plasmid is pHCMV-VSVG, pHCMV-VSVG-CMV-CymR or pHCMV-VSVG-CMV-TrpR.

[0086] 3. Dissolve the transfection reagent in the Op...

Embodiment 2

[0119] Example 2 Selection of the repressive operator

[0120] In the present invention, four kinds of transcription regulation systems are tested: CymR-CuO, tryptophan operator, diphtheria toxin inhibitor regulation system, and lactose operator.

[0121] 1. First, by introducing the cis-acting elements of these four systems on the lentiviral vector, test whether there is an effect of inhibiting the expression of foreign genes in the eukaryotic system.

[0122] 1. CymR-CuO system

[0123] CymR-CuO system is a kind of regulated expression system. The principle is as follows:

[0124] In the absence of Cumate (kulic acid), CymR protein can specifically bind to CuO elements, thereby inhibiting gene transcription. In the presence of Cumate, Cumate-bound CymR will dissociate from the CuO element, allowing normal gene expression.

[0125] The present invention constructs a series of promoter vectors of CMV-CuO, EF1a-CuO, SFH-CuO, and CAG-CuO, and confirms that the CuO element co...

Embodiment 3

[0143] Example 3 The repressor operator improves the virus titer of the lentiviral vector of the reverse expression cassette

[0144] If there is a reverse expression frame in the lentivirus, because the reverse expression frame exists, the viral RNA is rarely transcribed, so the virus titer is very low.

[0145] The construction of pSLenti-TK PolyA-mCherry-CMV-SFH-EGFP-P2A-Puro-WPRE ( Figure 17 ), pSLenti-TK PolyA-mCherry-CuO-CMV-SFH-EGFP-P2A-Puro-WPRE ( Figure 18 ), pSLenti-TK PolyA-mCherry-TrpO-CMV-SFH-EGFP-P2A-Puro-WPRE ( Figure 19 ) vectors, using CMV-CuO and CMV-TrpO promoter reverse expression cassettes, using CymR or TrpR and tryptophan in lentiviral packaging, together with the expression of inhibitory proteins, can effectively solve the problem of low toxicity of reverse expression cassettes (The fluorescence image of the experimental results is shown in Figure 20 As shown, the lentivirus titer chart is as follows Figure 21 shown).

[0146] This expression ...

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Abstract

The invention relates to the technical field of biology, in particular to a lentivirus packaging vector and a packaging method. The lentiviral packaging vector is provided with a first LTR located atthe upstream and a second LTR located at the downstream in the expression direction of a viral genome, wherein a reversely inserted gene expression cassette is arranged between the first LTR and the second LTR; the gene expression cassette comprises a promoter, a repression type operator, a target gene and a polyadenylation signal which are connected in sequence; and when a repression object exists, the repression type operator represses the expression of the target gene.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a lentiviral packaging vector and a packaging method. Background technique [0002] Lentivirus is a kind of viral vector modified from human immunodeficiency virus (HIV), and it is a kind of retrovirus. The genome is RNA, and its toxic gene has been deleted and replaced by an exogenous target gene. A pseudotyped virus. Reverse transcriptase can be used to integrate foreign genes into the genome to achieve stable expression, and it has the characteristics of infecting dividing and non-dividing cells. [0003] After the lentiviral genome enters the cell, it is reverse-transcribed into DNA in the cytoplasm to form a pre-DNA integration complex. After entering the nucleus, the DNA is integrated into the cell genome. The integrated DNA is transcribed into mRNA, which returns to the cytoplasm to express the target protein; or produces small RNA. Lentivirus-mediated gene expression or sm...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10
CPCC12N15/86C12N2740/15043C12N2830/50C12N2740/16043C12N2830/005C12N2740/16052C12N2740/15022C12N2740/15052
Inventor 杨兴林杨佳丽杨蕊菊潘讴东贾国栋夏清梅
Owner OBIO TECH SHANGHAI CORP LTD
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