Peste des petits ruminants virus (PPRV) reverse genetic operating system and application thereof

A technology of Peste des petits ruminants and an operating system, applied in the field of virus genetic manipulation, to achieve cost savings, significant social and economic benefits, and reduce stress responses

Active Publication Date: 2011-05-25
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] However, the reverse genetic manipulation technology of PPRV is still blank in the world. Therefore, this study aims to establish a PPRV reverse genetic manipulation technology platform to rescue PPRV virus and PPRV recombinant virus with recombinant EGFP protein in vitro, so as to further develop PPRV vector vaccine and PPRV Basic Research Provides Technology Platform

Method used

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  • Peste des petits ruminants virus (PPRV) reverse genetic operating system and application thereof
  • Peste des petits ruminants virus (PPRV) reverse genetic operating system and application thereof
  • Peste des petits ruminants virus (PPRV) reverse genetic operating system and application thereof

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Experimental program
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Effect test

Embodiment 1

[0044] Embodiment 1 Construction of Peste des petits ruminants virus reverse genetics operating system

[0045] 1 Materials and methods

[0046] 1.1 Materials

[0047] PPRV Nigeria75 / 1 (PPRV / N75 / 1) vaccine strain was purchased from China Veterinary Drug Administration; BHK-21 cells (ATCC No.CCL-10) and Vero cells (ATCC No.CCL-81) were purchased from Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences The zoonotic diseases research laboratory is preserved, and the culture medium is DMEM containing 10% fetal bovine serum; plasmid pCI is purchased from Promega, pBluescript and pIRES-EGFP are purchased from Clontech; PrimeSTAR HS DNA polymerase, T4 DNA ligase and others Restriction enzymes were purchased from TaKaRa Company; RNA extraction reagent Trizol, mouse reverse transcriptase (MLV), fetal calf serum, DMEM and calcium phosphate transfection kit (Calcium phosphate Transfection Kit) were purchased from Invitrogen; mouse anti- PPRV whole virus hyp...

Embodiment 2

[0087] Example 2 Recombinant virus forms CPE characteristics and GFP expression detection

[0088] The wtPPRV / N75 / 1, rPPRV / N75 / 1, rPPRV / 41EGFP and rPPRV / 101EGFP viruses prepared in Example 1 were inoculated at a MOI of 0.01 and grown overnight at a density of about 70% to 80% monolayer Vero cells at 37°C After 1 hour of infection, add DMEM culture solution containing 2% fetal bovine serum, in 5% CO2 , Culture at 37°C. The formation of CPE and the expression of EGFP protein were observed at 3, 5, and 7 days after infection.

[0089] In order to accurately compare the EGFP expression ability of rPPRV / 41EGFP and rPPRV / 101EGFP, the two strains of viruses were inoculated into Vero cells according to the above method. Add cell lysate (0.15mol / L Tris-Cl, pH 8.0, 1.5% Triton X-100, add 100 μl lysate for every 200 μl culture medium) at 3, 4, 5, 6 days after infection respectively, and after 15 minutes of lysis, Take 100 μl and add it to a white opaque 96-well plate (Corning), set the...

Embodiment 3

[0092] Embodiment 3 Recombinant virus compares with the growth kinetics characteristic of parent virus strain on Vero cell

[0093] Inoculate wtPPRV / N75 / 1, rPPRV / N75 / 1, rPPRV / 41GFP, and rPPRV / 101GFP at an MOI of 0.01 on single-layer Vero cells grown overnight at a density of approximately 70% to 80%, and inoculate at 37°C for 1 hour , add DMEM complete medium containing 2% fetal bovine serum, in 5% CO 2 , Culture at 37°C. The virus was harvested at 3, 4, 5, 6, 7 and 8 days after infection, and after freezing and thawing twice, the virus was diluted 10 times, and TCID was titrated on Vero cells in a 96-well plate 50 (half tissue infection amount), observe CPE or fluorescence under a fluorescent inverted microscope on the 14th day after infection, and calculate TCID 50 , to evaluate the growth kinetics of each virus on Vero cells.

[0094] The kinetic growth curves of wtPPRV / N75 / 1, rPPRV / N75 / 1, rPPRV / 41GFP and rPPRV / 101GFP on Vero cells are as follows Figure 5 As shown, the...

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Abstract

The invention relates to a peste des petits ruminants virus (PPRV) reverse genetic operating system and an application thereof. The PPRV reverse genetic operating system comprises a transcription plasmid and one or more helper plasmids, wherein the transcription plasmid can express the genome full-length cDNA (complementary deoxyribonucleic acid) sequence of the PPRV; and the helper plasmid(s) can express nucleoprotein (N), phosphoprotein (P) and polymerase large protein (L) of the PPRV, and virus replication-permitting host cells of the PPRV. By using the PPRV reverse genetic operating system, the recombined PPRV is successfully saved. The establishment of the PPRV reverse genetic operating technical platform provides an excellent technical platform for the development of PPRV live vector vaccines and the fundamental research related to PPRV.

Description

technical field [0001] The invention relates to the field of virus genetic manipulation, more specifically, the invention relates to a reverse genetic operating system of Peste des petits ruminant virus and its application. Background technique [0002] Peste des petits ruminants (PPR) is a highly contagious disease caused by Peste des petits ruminants virus (PPRV) [1]), which seriously endangers goats, sheep, and wild small ruminants. Animals such as ruminants [2] have even caused buffaloes to become ill [3]. [0003] The lethality rate of PPRV is 70-80%. In the new OIE classification, it is listed as an important economic animal disease, and the outbreak must be reported to OIE. PPR has been endangered and prevalent in Africa, the Middle East, Central Asia, South Asia and Southeast Asia for a long time. Pakistan[4] and Tajikistan[5] bordering on the west of my country have had epidemic outbreaks in recent years. In 2007, PPRV was introduced into the western border areas ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/62C12N7/08
Inventor 步志高陈伟业
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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