Reverse genetic operation system of Newcastle disease virus Mukteswar medium-toxicity vaccine strain and application of reverse genetic operation system

A technology of Newcastle disease virus and operating system, applied in the field of reverse genetic operating system of virulent vaccine strains, can solve the problems of reducing the rescue efficiency of Newcastle disease virus, long cycle, and reducing the ability of T7 RNA polymerase.

Active Publication Date: 2014-12-10
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem with this kind of reverse genetics operating system is that it is necessary to prepare a cell line stably expressing T7RNA polymerase. The process of preparing the cell line is complicated, the technical requirements are high, the cycle is long, and the antibiotics for maintaining the pressure need to be continuously added during the culture of the cell line. In order to maintain its stable expression of T7RNA polymerase, but with the increase of cell passage times, its ability to express T7RNA polymerase will continue to decrease until it disappears; in addition, the recombinant poxvirus expressing T7RNA polymerase infected during the transfection process will always Exist in cells, not only need to add arabinoside to inhibit the replication of the poxvirus in the process of rescuing the recombinant Newca

Method used

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  • Reverse genetic operation system of Newcastle disease virus Mukteswar medium-toxicity vaccine strain and application of reverse genetic operation system
  • Reverse genetic operation system of Newcastle disease virus Mukteswar medium-toxicity vaccine strain and application of reverse genetic operation system
  • Reverse genetic operation system of Newcastle disease virus Mukteswar medium-toxicity vaccine strain and application of reverse genetic operation system

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Embodiment 1

[0031] The construction of embodiment 1 Newcastle disease virus Mukteswar strain reverse genetics operating system

[0032] 1. Source of materials

[0033] Newcastle disease virus Mukteswar strain was purchased from Harbin Veken Biotechnology Development Company; plasmid pCI was purchased from

[0034] PROMEGA Company, pBluescript II ks(+ / -) Phagemids were purchased from Shanghai Jierui Biotechnology Co., Ltd.; DNA polymerase was purchased from TAKARA Company, T4DNA ligase and other restriction enzymes were purchased from NEB Company; viral RNA extraction reagents The kit and medium preparation kit were purchased from QIAGEN Company, and the M-MLV reverse transcription kit and liposome 2000 kit were purchased from IVENTROGEN Company.

[0035] 2. Method

[0036] 2.1 Construction of the transcription plasmid containing the whole genome cDNA sequence of Newcastle disease virus Mukteswar strain

[0037] In order to establish the reverse genetic operating system of Newcastle dis...

Embodiment 2

[0042] Example 2 Rescue of recombinant virus using the reverse genetic operating system constructed in Example 1

[0043] 1. Method:

[0044] The Vreo cells were inoculated in a 6-well cell culture plate, and the transfection was started when the cell monolayer grew to about 60%-80%. Wash the cells twice with serum-free DMEM medium, and then add Opti-MEM, 2ml / well; the transfection reagent is Liposome 2000 kit from Invitrogen Company, and the transfection is carried out according to the kit instructions, and the transcribed plasmid pCI-NDV and The helper plasmids pCI-NP, pCI-P, and pCI-L were transfected at a mass ratio of 4:2:2:1, with an amount of 4ug per well. After 6h to 8h of transfection, discard the transfection supernatant , add DMEM containing 5% FBS, when the transfected cells are almost all dead, absorb the supernatant to inoculate Vero cells in a new culture flask, and then observe the cytopathic changes day by day. When there is a significant syncytia formation, ...

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Abstract

The invention relates to a reverse genetic operation system of a Newcastle disease virus Mukteswar medium-toxicity vaccine strain and an application of the reverse genetic operation system. The system comprises a transcription plasmid and three transcription auxiliary plasmids, wherein the transcription plasmid comprises a whole genome cDNA sequence of the Newcastle disease virus Mukteswar medium-toxicity vaccine strain which is controlled by an eukaryotic promoter; the three transcription auxiliary plasmids are controlled by the eukaryotic promoter; and each transcription auxiliary plasmid comprises a cDNA sequence which is used for encoding the nucleoprotein (NP) of the Newcastle disease virus Mukteswar medium-toxicity vaccine strain, a cDNA sequence which is used for encoding the phosphoprotein (P) of the Newcastle disease virus Mukteswar medium-toxicity vaccine strain and a cDNA sequence which is used for encoding the large polymerase (L) of the Newcastle disease virus Mukteswar medium-toxicity vaccine strain. According to the reverse genetic operation system, the wild type recombined Newcastle disease virus is successfully rescued, and a foundation is laid for the further development of the vaccine development by taking the Newcastle disease virus as a carrier and the fundamental researches related to the tumor oncolytic treating virus and the NDV (Newcastle Disease Virus).

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 Newcastle disease virus Mukteswar intermediate virulence vaccine strain and its application. Background technique [0002] Angela Romer-Oberdo rfer et al. and BEN P.H.PEETERS et al. first published the articles "Generation of recombinant lentogenic Newcastle disease virus from cDNA" (Journal of General Virology (1999), 80, 2987–2995) and "Rescue of Newcastle Disease Virus" in 1999, respectively. from Cloned cDNA: Evidence that Cleavability of the Fusion Protein Is a Major Determinant for Virulence" (JOURNAL OF VIROLOGY, June 1999, p.5001–5009), discloses a transcription system controlled by the T7 promoter to rescue attenuated Newcastle disease virus Afterwards, some people rescued different strains of Newcastle disease virus one after another. The rescue systems of all these Newcastle disease viruses are c...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N7/01A61K39/17A61P31/14C12R1/93
Inventor 王永
Owner HARBIN MEDICAL UNIVERSITY
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