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

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...

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 Newcastle disease virus, but also need to remove the poxvirus by filtration or multiple dilution and passage after the rescue of the recombinant virus, which not only It will reduce the rescue efficiency of Newcastle disease virus, and there is also the risk of poxvirus contamination. In the research of this method, if the transfected cell culture is not filtered, the rescue efficiency of Newcastle disease virus is high, but the removal of poxvirus is very difficult. Complicated, Newcastle disease virus rescue is inefficient or fails if transfected cell cultures are filtered

[0004] The present invention overcomes the shortcoming that specific cell lines and recombinant poxvirus must provide T7 RNA polymerase in the prior art, and provides a method that can use multiple cell lines to rescue recombinant Newcastle disease virus

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