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Infectious clone containing rabies virus glycoprotein and application

A technology for infectious cloning and rabies virus, which is applied to infectious cloning and application fields containing rabies virus glycoprotein, can solve problems such as unclear mechanism, and achieve the effects of rapid proliferation, high success rate and simple operation

Active Publication Date: 2021-08-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] To date, the mechanism by which infectious VLVs form is unclear

Method used

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  • Infectious clone containing rabies virus glycoprotein and application
  • Infectious clone containing rabies virus glycoprotein and application
  • Infectious clone containing rabies virus glycoprotein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Obtaining of an infectious clone containing rabies virus glycoprotein:

[0030] 1) Construction of Semliki forest virus replicon driven by CMV promoter: pSF V3 (purchased from addgene) was transformed by using multi-segment homologous recombination technology, the original SP6 promoter was replaced by CMV promoter, and multimer Insert the hepatitis D virus ribozyme sequence (HdvRz) and bovine auxin polyadenylation signal sequence (bGH poly(A) signal) behind the adenine tail, eliminating the step of in vitro transcription, the specific method is as follows:

[0031] 4 pairs of homologous recombination primers were designed (TY-A-F / R, TY-B-F / R, TY-C-F / R, TY-D-F / R in Table 1), wherein TY-A-F / R is to amplify HdvRz and bGH The primers for poly(A) signal, TY-C-F / R are the primers for amplifying CMV promoter (the template plasmid BAC containing CMV promoter, HdvRz and bGH poly(A) signal was kindly provided by Professor Peng Guiqing of Huazhong Agricultural University, Wang G ...

Embodiment 2

[0053] The rabies virus-like vesicles VLVs-P50R, VLVs-P50R-P25 and VLVs-P50R-P25R virus fluorescent spot diffusion and growth curve determination provided by the invention:

[0054] Fluorescent spot diffusion assay of virus-like vesicles: mix 1 x 10 5 BHK-21 cells were inoculated into each well of a 24-well plate, and when the confluence of the cells reached 90%, VLVs-P50R, VLVs-P50R-P25 or VLVs-P50R-P25R were inoculated at MOI=0.001, 37°C, 5% CO 2 After adsorbing in the incubator for 1 h, wash with PBS three times, add 2% FBS-containing DMEM medium and 2% methylcellulose cover and store at 37°C, 5% CO 2 Cultured in an incubator, took out a plate at 24h and 48h respectively, discarded the cover, washed three times with PBS, fixed with pre-cooled 80% acetone at -20°C for 20min, and then observed the formed cells by indirect immunofluorescence. Fluorescent spots ( image 3 Middle A). In the experimental group, 5 fluorescent spots were randomly selected in each well, and the p...

Embodiment 3

[0057] Purification, SDS-PAGE and Western Blot of rabies virus-like vesicles VLVs-P50R, VLVs-P50R-P25, VLVs-P50R-P25R provided by the invention:

[0058] Purification and SDS-PAGE of VLVs-P50R, VLVs-P50R-P25 and VLVs-P50R-P25R: Ultracentrifuge the collected VLVs-P50R, VLVs-P50R-P25 and VLVs-P50R-P25R at 30,000rpm at 4°C for 2.5 h (Beckman, SW32Ti), discard the supernatant, add 1ml PBS to the bottom of the tube, let it dissolve overnight at 4°C, take 100μl, add 5×SDS loading buffer and boil it for SDS-PAGE detection, and at the same time use purified LBNSE The virus is used as a marker, and the rest of the concentrated virus is subjected to 20%-60% sucrose density gradient centrifugation, and a precipitate layer appears at a sucrose concentration of 40%. Carefully absorb this layer of precipitate, add PBS to dilute, and perform ultra-high-speed centrifugation again to remove sucrose, and finally use 100ul PBS was dissolved at 4°C overnight, and frozen at -80°C for later use. T...

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Abstract

The invention belongs to the technical field of biology, and particularly discloses infectious clone containing rabies virus glycoprotein and application of the infectious clone. The infectious clone is constructed by a method that on the basis of a Semliki forest virus vector, an SP6 promoter sequence in the Semliki forest virus vector is replaced by a CMV promoter sequence, a hepatitis D virus ribozyme sequence (HdvRz) and a bovine auxin polyadenylation signal sequence are inserted behind a polyadenylate tail sequence, 13 base mutations are introduced into a non-structural protein coding region, and finally, rabies virus glycoprotein coding sequences are inserted at multiple cloning sites. The rabies virus-like vesicles rescued by the reverse genetic manipulation system only have one kind of structural protein RABV-G, proliferation passage can be carried out on BHK-21 cells, the titer is as high as 108 FFU / ml, and the rabies virus-like vesicles can be used as ideal rabies candidate vaccine strains.

Description

technical field [0001] The invention belongs to the technical field of biotechnology, and in particular relates to an infectious clone containing rabies virus glycoprotein and its application. Background technique [0002] Rabies is a zoonotic infectious disease caused by Rabies virus (RABV) and characterized by central nervous system infection. For rabies, there is no specific treatment or medicine, but vaccination can effectively prevent the occurrence and spread of rabies. [0003] The rabies vaccines that have been studied more at present mainly include attenuated vaccines, inactivated vaccines, genetically engineered vaccines and recombinant vaccines constructed by reverse genetic technology. Among them, attenuated vaccines and inactivated vaccines are widely used. Attenuated vaccines are mainly used in developed countries such as Europe and the United States for intramuscular injection or oral immunization of wild animals and domestic animals in developing countries....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N15/86A61K39/205A61P31/14C12R1/93
CPCC12N7/00C12N15/86A61K39/12A61P31/14C12N2760/20134C12N2760/20152C12N2770/36143C12N2800/107C12N2800/22
Inventor 赵凌张程光陈晨田玉玲赵雨欣
Owner HUAZHONG AGRI UNIV
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