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Exogenous gene controllable expression adenovirus packaging method

A gene expression cassette, adenovirus technology, applied in the field of molecular biology, can solve the problems of inability to obtain virus particles and little attention to the impact

Active Publication Date: 2021-03-16
OBIO TECH SHANGHAI CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, research on viral vectors is focused on infectivity, specificity, packaging capacity, etc., but little attention has been paid to the impact of the packaged foreign gene (gene of interest, GOI) on the virus. In practice, a considerable part of the foreign gene Genes are unable to obtain effective virus particles

Method used

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  • Exogenous gene controllable expression adenovirus packaging method
  • Exogenous gene controllable expression adenovirus packaging method
  • Exogenous gene controllable expression adenovirus packaging method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] The selection of embodiment 1 repressor type operator

[0069] In the present invention, four kinds of transcription regulation systems are tested: CymR-CuO, tryptophan operator, diphtheria toxin inhibitor regulation system, and lactose operator.

[0070] First, by introducing the cis-acting elements of these four systems on the lentiviral vector (the vector is a shuttle vector, which can be used as a general-purpose eukaryotic vector for transfection of cells alone, and its conclusion is not limited to lentivirus), it is tested whether it is in true In the nuclear system, it has the effect of inhibiting the expression of foreign genes.

[0071] 1. Plasmid transfection

[0072] The method for plasmid transfection 293T specifically includes:

[0073] 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 i...

Embodiment 2

[0099] Example 2 ADV virus packaging and virus titer detection

[0100] 1. Construction of HEK-293-CMV-CymR and HEK-293-CMV-TrpR stable cell lines

[0101] This embodiment constructed HEK-293-CMV-CymR and HEK-293-CMV-TrpR stable cell lines, specifically including:

[0102] Use MfeI and BstBI (NEB Company) to linearize pcDNA3.1-CymR or pcDNA3.1-TrpR by double enzyme digestion; after 4 to 6 hours of enzyme digestion, use a recovery kit (QIAGEN Company) to recover the linearized band After the recovery is completed, measure the concentration and confirm with the agarose gel;

[0103] The day before plasmid transfection, HEK 293 cells in good condition were selected, collected after digestion, and counted according to 2×10 5 Spread each well on a 24-well plate, shake well, and place at 37°C, 5% CO 2 Incubator cultivation. Change medium before transfection: 1 hour before plasmid transfection, take out the 24-well plate laid out the day before from the incubator, discard the ori...

Embodiment 3

[0139] Example 3 ADV virus vector expresses CMV-NLS-iCre gene

[0140] In the virus production, we found that the virus particles obtained by constructing the CMV-NLS-iCre gene into the common ADV vector could not be effectively amplified. The use of CMV-CuO and CMV-TrpO promoters in conjunction with stable strains that inhibit protein expression can effectively solve the problem of toxicity ( Figure 17 , Figure 18 ).

[0141] Plasmids pShuttle-CMV-iCre-3×Flag-P2A-sfGFP, pShuttle-CMV-TrpO-iCre-3×Flag-P2A-sfGFP and pShuttle-CMV-CuO-iCre-3×Flag-P2A used in this example The plasmid map of -sfGFP is as follows Figure 19 , Figure 20 , Figure 21 shown.

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Abstract

The invention relates to the field of molecular biology, and particularly relates to an exogenous gene controllable expression adenovirus packaging vector. The adenovirus packaging vector adopted by the method has two ITR fragments and a gene expression cassette inserted between the two ITR fragments; the gene expression cassette comprises a promoter, a repressor operator and an optional target gene which are connected in sequence; and when a repressor exists, the repressor operator can repress the expression of the downstream target gene. The repressor operator is creatively integrated into the adenovirus vector, so that the exogenous gene expression in the virus packaging process can be reduced, the influence of the exogenous gene is eliminated, and the yield of specific viruses is increased.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an adenovirus packaging method for controllable expression of exogenous genes. Background technique [0002] Viral vectors can bring genetic material into cells. The principle is to use the molecular mechanism that viruses have high affinity to bind to cell surface receptors, and transmit their genomes into certain types of cells for infection. Widely used in basic research, gene therapy or vaccines. [0003] Adenoviruses are particularly suitable for use as viral vectors. Adenovirus is a non-enveloped virus with a diameter of about 90nm to 100nm, including a nucleocapsid and a linear double-stranded DNA genome. Viral nucleocapsids include penton capsids and hexon capsids. A unique fiber is associated with each penton matrix and facilitates the attachment of the virus to the host cell via the coxsackievirus-adenovirus receptor on the host cell surface. More than 50 strains of...

Claims

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Application Information

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IPC IPC(8): C12N15/861
CPCC12N15/86C12N2710/10343C12N2710/10352C12N2800/107C12N2830/005Y02A50/30
Inventor 杨兴林杨佳丽杨蕊菊贾国栋由庆睿
Owner OBIO TECH SHANGHAI CORP LTD