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Method for improving transduction efficiency of rhabdovirus on mammalian cells

A mammalian and baculovirus technology, applied in the field of improving the transduction efficiency of baculovirus to mammalian cells, can solve the problems of time-consuming and complicated operation, and achieve the effect of prolonging the experiment time and making the overall method simple and easy to operate

Inactive Publication Date: 2011-09-28
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires a high titer of virus to achieve a relatively ideal transduction efficiency. In the early stage, a large number of viruses need to be amplified and preparation processes such as ultracentrifugation are required. The operation is complicated and time-consuming.
In recent years, there have also been reports of improving transduction efficiency by displaying foreign proteins on the surface of the virus, but this requires additional genetic manipulation of the virus, and there is currently no simple and rapid method to improve the transduction efficiency of AcMNPV

Method used

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  • Method for improving transduction efficiency of rhabdovirus on mammalian cells
  • Method for improving transduction efficiency of rhabdovirus on mammalian cells
  • Method for improving transduction efficiency of rhabdovirus on mammalian cells

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1. Method for improving transduction efficiency of baculovirus to mammalian cells

[0037] This method includes the combination of virus and cell at low temperature (2-6°C), short-term induction of low pH (pH 4.4-5.6) after virus binding, superposition of endocytic pathway inhibitor and low pH induction. Specifically, a method including the following steps is adopted.

[0038] 1. Preparation:

[0039] (1) Relevant reagents: Lipofectin was purchased from invitrogen; Grace’s insect medium, pH 6.0 was purchased from Gibco; fetal calf serum was purchased from Gibco; DMEM medium, pH=7.0 was purchased from Gibco; ammonium chloride was purchased from Beijing Perseverance Company.

[0040] (2) Instruments used: flow cytometer, ultra-clean bench, fluorescence microscope, centrifuge, etc.

[0041] 2. Construction of the virus:

[0042] use Bam HI was cut from pFB-Op166 plasmid Orgyia pseudotsugata MNPV (OpMNPV) gp64 promoter Op166 and inserted into plasmid pEGFP-N1 to construct ...

Embodiment 2

[0057] Example 2. Use AcMNPV as a vector for expression of foreign proteins

[0058] The method of the invention combined with sodium butyrate is used for AcMNPV as a carrier to express foreign proteins.

[0059] This method includes the combination of virus and cells at low temperature (2-6°C), short-term induction of low pH (pH 4.4-5.6) after virus binding, low pH induction and sodium butyrate overlap. Specifically, a method including the following steps is adopted.

[0060] 1. Preparation:

[0061] (1) Related reagents: Lipofectin was purchased from invitrogen; Grace’s insect medium, pH 6.0 was purchased from Gibco; fetal bovine serum was purchased from Gibco; DMEM medium, pH=7.0 was purchased from Gibco; sodium butyrate was purchased from Sigma.

[0062] (2) Instruments used: ultra-clean workbench, fluorescence microscope, centrifuge, semi-dry transfer membrane instrument, etc.

[0063] 2. Construction of the virus:

[0064] use Sal I and Not I cut the CAG-HA with mammalian CAG pr...

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Abstract

The invention relates to a method for improving the transduction efficiency of rhabdovirus on mammalian cells. The method comprises the following steps of: preparing, constructing viruses, amplifying the viruses, determining titer, and performing low-pH-induced transduction, wherein the low-pH-induced transduction comprises the steps of pretreating cells and the viruses, combining the viruses with the cells, and performing low-pH-induced membrane fusion. The method is combined with sodium butyrate, so that AcMNPV serving as a vector expresses an exogenous protein through the steps of pretreating the cells and the viruses, combining the viruses with the cells, and performing low-pH-induced membrane fusion. By the method, the transduction efficiency of the AcMNPV on the mammalian cells and the expression level of the exogenous protein can be remarkably improved; and the method has potential application valve in the expression of exogenous proteins and gene therapy.

Description

Technical field [0001] The present invention relates to the technical field of virus transduction, in particular to a method for improving the transduction efficiency of baculovirus to mammalian cells. Background technique [0002] Baculovirus (AcMNPV) can transduce mammalian cells through the endocytic pathway. Under the acidic environment of the endosome, the membrane fusion protein GP64 on the surface of the viral envelope induces the fusion of the viral envelope and the endosomal membrane and the virus nucleus The capsid is released into the cytoplasm and finally completes the transduction. AcMNPV transduction can be widely used in the introduction and expression of foreign genes. [0003] The current transduction technology is mainly based on high-titer virus and cell incubation. This method requires a high-titer virus to achieve a more ideal transduction efficiency. In the early stage, a large number of viruses need to be amplified and preparation processes such as ultracen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866
Inventor 王华林董思聪王曼丽沈姝邓菲胡志红
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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