Vesicular stomatitis virus glycoprotein variant and construction method and application thereof

A technology of protein variants and membrane proteins, applied in the direction of viruses/bacteriophages, applications, viruses, etc., can solve the problems of virulence recovery and inapplicability, and achieve the effects of reducing infectivity, good safety, and reducing fusion ability

Inactive Publication Date: 2019-12-17
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, attenuated vaccines also have certain defects. For example, a large number of long-term screenings are required for the strains, the virulence may be restored, and the virus can proliferate in the body, so it may not be suitable for immunocompromised persons.

Method used

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  • Vesicular stomatitis virus glycoprotein variant and construction method and application thereof
  • Vesicular stomatitis virus glycoprotein variant and construction method and application thereof
  • Vesicular stomatitis virus glycoprotein variant and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1 Experiment related methods and processes

[0070] 1.1. Cell culture

[0071] HEK293T( CRL-11268) and Hela ( CRM-CCL-2) cells were cultured at 37°C, saturated humidity, 5% CO 2 in the cell culture incubator.

[0072] 1.2. Western blot

[0073] 1) Discard the culture medium and wash twice with PBS buffer;

[0074] 2) Add pre-cooled whole-cell protein lysate (50mM Tris (pH 7.4), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate), about 30μL / mL cells, pipette to mix, place on ice for 10min ;

[0075] 3) Centrifuge the cell lysate suspension at 13300 rpm at 4°C for 10 min, transfer the supernatant to a new EP tube, and obtain the cell protein extract;

[0076] 4) Add 6x SDS loading buffer (6x loading buffer: 3.75mL 1M Tris pH 6.8, 1.2g SDS, 6mL glycerol, 0.006g bromophenol blue, 0.462g DTT) in proportion, and bathe in boiling water at 100°C for 5-10min;

[0077] 5) Load the sample on the SDS-PAGE gel. When the sample is in the stacking gel, electrophore...

Embodiment 2V

[0121] Cell fusion mediated by embodiment 2VSV-G

[0122] As a viral fusion protein, VSV-G's fusion ability has been confirmed. In order to quantitatively analyze its fusion function and monitor the kinetic process of fusion in real time, the inventors constructed a cell-cell fusion system. The red fluorescent protein (dsRED-nes) with nuclear export signal (dsRED-nes) and VSV-G were co-transfected into cells, the cytoplasm of the transfected cells was red under the fluorescence microscope, and VSV-G was expressed on the surface of the cell membrane; in addition, the cells with Cells were transfected with cyan fluorescent protein (CFP-nls) with nuclear localization signal, and the nuclei of the transfected cells were cyan under the fluorescence microscope. These two kinds of cells were co-cultured, and low pH was used to induce VSV-G-mediated fusion. If the cells with red cytoplasm and more than one cyan nucleus were fused cells, such as figure 1 and figure 2 shown.

[0...

Embodiment 3

[0148] Example 3 Preparation of Lentivirus Expressing VSV-G Variants and Cell Infection Experiment

[0149] As a viral fusion protein, VSV-G mediates the fusion of the viral envelope with the endocytic membrane of the target cell, and then invades the cell. The host range of VSV-G-mediated fusion is very wide. VSV-G can be expressed on the surface of genetically engineered lentivirus as a fusion agent of lentiviral transfection vector. Since the genetically engineered lentivirus has no self-replication ability, it is safe High reliability, the system has been widely used in research. In the lentiviral packaging system consisting of three plasmids, pLVTHM, pMD 2.G and psPAX2 (all purchased from Addgene), pMD 2.G carries the VSV-G gene, so that the obtained virus surface expresses VSV-G, which can mediate Fusion of virus and cells. The inventors used conventional molecular cloning techniques to modify VSV-G with reference to Example 2, replacing its transmembrane region or ins...

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Abstract

The invention relates to a vesicular stomatitis virus glycoprotein (VSV-G) variant and its construction method and its application in the preparation of vesicular stomatitis virus (VSV) vaccines.

Description

technical field [0001] The invention relates to a variant of vesicular stomatitis virus envelope glycoprotein (VSV-G) function loss and its construction method and application. Background technique [0002] Vesicular Stomatitis Virus / Vesicular Stomatitis Virus (VSV) is a member of the rhabdovirus family and belongs to enveloped viruses. This type of virus is named for its bullet-like shape. Its family members include VSV and rabies virus and passenger viruses. [0003] VSV can infect a variety of animals and insects. Domestic animals that are naturally infected with VSV include horses, cattle, sheep and pigs, and wild animals (wild boars, raccoons and deer, etc.) can be naturally infected. Infected animals present with fever, lethargy, decreased appetite, vesicular lesions on the mouth, teats, between the toes, and on the crests of the hooves, and pot-shaped rings on the legs. The blisters are easy to rupture, exposing granulation tissue, showing red erosion, surrounded b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/145C12N15/47A61K39/205A61P31/14
CPCA61K39/12A61K2039/5254A61K2039/552A61P31/14C07K14/005C12N2760/20222C12N2760/20234
Inventor 石磊慈雅丽
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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