Construction of rabies virus G protein expression recombinant canine distemper virus CDV/R-20/8 vaccine strain

A canine distemper virus, rabies virus technology, applied in the direction of virus/phage, recombinant DNA technology, antiviral agent, etc., can solve the problems of limited wide application, complex production process of cell vaccine, high price and so on

Active Publication Date: 2012-02-08
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The production process of cell seedlings is complicated and expensive, which also limits the wide application in production practice

Method used

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  • Construction of rabies virus G protein expression recombinant canine distemper virus CDV/R-20/8 vaccine strain
  • Construction of rabies virus G protein expression recombinant canine distemper virus CDV/R-20/8 vaccine strain
  • Construction of rabies virus G protein expression recombinant canine distemper virus CDV/R-20/8 vaccine strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The construction of embodiment 1 canine distemper virus reverse genetics operating system

[0043] 1 Materials and methods

[0044] 1.1 Materials

[0045] The attenuated canine distemper vaccine CDV / R-20 / 8 strain was purchased from the Military Veterinary Research Institute of the Academy of Military Medical Sciences of the PLA; BHK-21 cells (suckling hamster kidney cells ATCC No.CCL-10) and Vero cells (African green monkey kidney cells, ATCC No.CCL-81), the culture medium is DMEM containing 10% fetal calf serum; plasmid pCI is purchased from Promega, pBluescript is purchased from Clontech; PrimeSTAR (R) HS DNA polymerase, T4 DNA ligase and other restriction enzymes All were purchased from TaKaRa Company; RNA extraction reagent Trizol, mouse-derived reverse transcriptase (MLV), fetal bovine serum and calcium phosphate transfection kit (Calcium phosphate Transfection Kit) were purchased from Invitrogen; mouse anti-canine distemper virus whole virus high The immune seru...

Embodiment 2

[0066] The growth kinetics comparison of embodiment 2 recombinant virus and wild-type virus on Vero cells

[0067] The wild-type virus rCDV and the recombinant virus rCDV-EGFP prepared in Example 1 were respectively inoculated at an MOI of 0.01 on single-layer Vero cells that grew overnight and had a density of about 70% to 80%. % FBS in DMEM complete medium, 5% CO 2 , cultured at 37°C, and the cells infected with the above virus were harvested at 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h after infection; the recombinant virus rCDV-EGFP harvested at different time periods and the parent strain virus liquid were frozen and thawed After 1 time, make 10-fold serial dilutions, take 100 μL of the virus solution of each dilution and inoculate them on a 96-well plate, grow overnight, and the density is about 70% to 80% monolayer Vero cells, incubate at 37°C for 1 hour, wash with PBS 2 times, add DMEM complete culture medium containing 5% fetal bovine serum, 5% CO 2 , cultured at 37...

Embodiment 3

[0069] Example 3 Exogenous protein EGFP expression detection

[0070] In order to evaluate the stability of recombinant virus rCDV-EGFP expressing GFP in Vero cells, 5 consecutive passages of recombinant virus rCDV-EGFP from F1 to F5 were inoculated at a MOI of 0.01 in a monolayer of Vero cells grown overnight at a density of about 70% to 80%. Cells were incubated at 37°C for 1 hour, then added with DMEM complete culture medium containing 5% fetal bovine serum, 5% CO 2 , cultured at 37°C, and observed the results under a fluorescence microscope (Leica DMIRES2) after 3 days. The results show that all generations of recombinant virus rCDV-EGFP can express GFP efficiently in Vero cells, see Figure 4 . Construction and biological activity of the recombinant canine distemper virus vaccine expressed in the gene of embodiment 4 encoding rabies virus glycoprotein (G protein)

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Abstract

The invention relates to a recombinant canine distemper virus capable of expressing a rabies virus glycoprotein (G protein). Specifically, the recombinant canine distemper virus is rCDV-RVG. The invention also discloses a method for preparing a vaccine strain from the recombinant canine distemper virus, and a use of the recombinant canine distemper virus in preparation of a bivalent vaccine for preventing rabies and canine distemper.

Description

technical field [0001] The invention relates to the field of recombinant virus vaccines, more specifically, the invention relates to a recombinant canine distemper virus vaccine expressing rabies virus glycoprotein (G protein), more specifically, the recombinant canine distemper virus vaccine is rCDV-RVG. The invention also discloses a method for preparing the recombinant canine distemper virus and the application of the recombinant canine distemper virus in preparing a dual vaccine for preventing rabies and canine distemper. Background technique [0002] Canine distemper (CD) is an acute, highly contagious and fatal infectious disease of canines (especially puppies), mustelidae and some raccoon animals caused by canine distemper virus (CDV) , can cause a large number of animals such as dogs, minks, and foxes to become ill, and even tigers, leopards, monkeys, and giant pandas can also be infected, and the fatality rate is different in different hosts [1] , the fatality rate...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/47C12N15/63C07K14/145A61K39/295A61P31/14C12R1/93A61K39/175A61K39/205
Inventor 步志高夏咸柱王喜军冯娜葛金英杨松涛
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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