High-stability pseudoviral particles as well as plasmid vector and method for preparing pseudoviral particles

Abstract

The invention provides high-stability pseudoviral particles as well as a plasmid vector and a method for preparing the pseudoviral particles. The pseudoviral particles contain a nucleic acid fragment of norovirus, wherein the nucleic acid fragment can serve as a target for detecting the norovirus. The plasmid vector contains a maturase coding gene of a Qbeta phage, a capsid protein coding gene, a packaging site sequence and a DNA segment including a cDNA sequence in correspondence with the nucleic acid fragment. The method comprises the following steps: constructing the plasmid vector, then transcribing and / or translating the plasmid vector in escherichia coli cells, and conducting separating and purifying, so that the pseudoviral particles are obtained. With the application of the method disclosed by the invention, the pseudoviral particles, which contain the norovirus nucleic acid fragment functioning as the detection target, can be prepared; and the pseudoviral particles have the advantages of being free from infectivity, high in copy number, good in stability and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a highly stable pseudovirion particle containing norovirus nucleic acid fragments and a plasmid vector and method for its preparation. Background technique [0002] Norovirus (Norovirus, NoV) was discovered in 1972. It belongs to the family Caliciviridae and the genus Norovirus. one. Norovirus exists stably in the environment, requires only a small infectious dose to cause disease, and can be transmitted through contaminated water, food, air, etc., so it often causes serious public health problems and food safety problems. According to the similarity of the RNA-dependent RNA polymerase and capsid protein coding gene sequences of Norovirus, Norovirus is divided into 5 genomes: GI, GII, GIII, GIV, and GV. [0003] RT-PCR method and real-time RT-PCR method have become the "gold standard" methods for norovirus detection due to their advantages of high sensitivity, good repe...

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Problems solved by technology

However, since norovirus cannot be cultured in vitro, molecular biology methods such as real-timeRT-PCR do not have a uniform standard sample as a positive control, and evaluation of the virus extraction process and detection results cannot be carried out.

[0004] Combining domestic and foreign references, my country's industry standards, and international standards, most of them recommend using positive diarrhea samples as positive standard samples, but diarrhea samples have the following two major disadvantages as positive standard samples: (1) The virus content is not uniform, and the virus content of different patients The virus concentration in diarrhea samples may vary by dozens or hundreds of times, and cannot be used in important links such as the sensitivity evaluation of detection methods and the comparison of test results in different laboratories; (2) There are serious biological safety hazards, and live viruses are positive In contrast, there is the possibility of operator infection and subsequent large-scale infection

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