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A lentiviral packaging method for the controllable expression of foreign genes

A technology of lentivirus packaging and gene expression cassettes, applied in the field of molecular biology, can solve the problems of reducing the infectivity of virus particles, affecting lentiviruses, etc., saving time, manpower and material resources, eliminating the influence of foreign genes, and increasing production. Effect

Active Publication Date: 2022-08-05
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, there are two NFκB binding sites on the LTR of HIV-1, and its transcription is heavily dependent on the activation of the canonical NFκB pathway, while the high expression of UBXN gene family members (UBXN1, N9 and N11) can block this pathway, thus greatly affecting Production of lentivirus
In addition, some genes will be expressed in the lentiviral envelope, such as MARCH8, although it will not reduce the production of lentivirus, but its overexpression will seriously reduce the infectious ability of viral particles

Method used

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  • A lentiviral packaging method for the controllable expression of foreign genes
  • A lentiviral packaging method for the controllable expression of foreign genes
  • A lentiviral packaging method for the controllable expression of foreign genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Packaging and titer detection of lentivirus

[0086] 1. Plasmid transfection

[0087] The method for plasmid transfection of 293T specifically includes:

[0088] 1. One day before transfection, inoculate 293T cells into the 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 A complex of staining reagent and plasmid, including the following steps:

[0089] 2. Mix and dissolve one or more plasmids to be transfected in Opti-MEM medium in equal proportions as required, mix gently, and let stand to obtain a plasmid dilution.

[0090] 3. Dissolve the transfection reagent in Opti-MEM medium, mix gently, and let it stand to obtain the transfection reagent diluent; add the transfection reagent diluent dropwise to the plasmid diluent, and mix gently while adding After 15-25min at room temperature, the DNA and the t...

Embodiment 2

[0129] Example 2 Selection of repressible operator

[0130] The present invention tests a total of 4 transcriptional regulation systems: CymR-CuO, tryptophan operator, diphtheria toxin inhibitor regulation system, and lactose operator.

[0131] 1. First, by introducing the cis-acting elements of these four systems into the lentiviral vector, test whether it has the effect of inhibiting the expression of foreign genes in the eukaryotic system.

[0132] 1. CymR-CuO system

[0133] CymR-CuO system is a kind of regulated expression system. The principle is as follows:

[0134] CymR protein can specifically bind to CuO element in the absence of Cumate (citric acid), thereby inhibiting gene transcription. In the presence of Cumate, the CymR bound to Cumate will detach from the CuO element, allowing the gene to be expressed normally.

[0135] The present invention constructs a series of promoter vectors of CMV-CuO, EF1a-CuO, SFH-CuO and CAG-CuO, which confirms that the CuO elemen...

Embodiment 3

[0153] Example 3 Expression of C1V1(t / t)-TS-mCherry gene by lentiviral vector

[0154] The proteins that combine the photosensitive channel-1 and the photosensitive channel (VChR1) discovered by Volvox are collectively referred to as C1V1. In virus production, we found that C1V1(t / t)-TS-mCherry was constructed into a common lentiviral vector and could not be packaged to obtain lentiviral particles.

[0155] By adopting the lentiviral packaging vector system of the present invention, packaging is carried out on the premise of having repressors, and the virus packaging titer can be effectively improved ( Figure 17 , Figure 18 ). The plasmids used in this example are pSLenti-CMV-C1V1(t-t)-TS-mCherry-3xFLAG-PGK-puro-WPRE, pSLenti-CMV-CuO-C1V1(t-t)-TS-mCherry-3xFLAG-PGK-puro-WPRE And the plasmid map of pSLenti-CMV-TrpO-C1V1(t-t)-TS-mCherry-3xFLAG-PGK-puro-WPRE is as follows Figure 19 , Figure 20 , Figure 21 shown.

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Abstract

The invention relates to the field of molecular biology, in particular to a lentivirus packaging method for controllable expression of foreign genes. The lentiviral packaging vector used in this method is provided with a first LTR located upstream and a second LTR located downstream along the expression direction of the viral genome, and there is a reversely inserted gene expression between the first LTR and the second LTR A cassette; the gene expression cassette comprises a sequentially connected promoter, a repressor-type operator and an optional target gene; when there is a repressor, the repressor-type operator can repress the expression of its downstream target gene. The invention creatively integrates the repressor-type operon into the lentiviral vector, which can reduce the expression of exogenous genes in the process of virus packaging, thereby eliminating the influence of exogenous genes and increasing the yield of specific viruses.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a lentivirus packaging method for controllable expression of foreign genes. Background technique [0002] Viral vectors can bring genetic material into cells. The principle is to use the molecular mechanism by which viruses bind to cell surface receptors with high affinity and transmit their genomes into certain types of cells for infection. Widely used in basic research, gene therapy or vaccines. [0003] Lentivirus vector is a gene therapy vector developed on the basis of HIV-1 (human immunodeficiency virus type I). Different from general retroviral vectors, it has the ability to infect both dividing and non-dividing cells. [0004] The research on lentiviral vectors has developed rapidly and the research is also very in-depth. The vector can effectively integrate the foreign gene into the host chromosome, so as to achieve persistent expression. In terms of infectivity, it ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867
CPCC12N15/86C12N2740/15043C12N2740/15052C12N2800/107C12N2830/005
Inventor 由庆睿杨兴林杨佳丽马佩敏贾国栋
Owner OBIO TECH SHANGHAI CORP LTD
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