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Norovirus RNA fragment-containing pseudoviral particle and preparation method thereof

A technology of pseudovirus particles and viruses, which is applied in the field of pseudovirus particles containing norovirus RNA fragments and its preparation, can solve the problems of missed detection and inability to detect virus strains, and achieve false negative avoidance, easy storage and transportation, and good The effect of stability

Inactive Publication Date: 2014-01-01
HENAN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, due to virus mutation and recombination, primers and probes for specific strains cannot detect all strains, often resulting in missed detection, so the design of primers and probes that can detect all strains is very critical

Method used

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  • Norovirus RNA fragment-containing pseudoviral particle and preparation method thereof
  • Norovirus RNA fragment-containing pseudoviral particle and preparation method thereof
  • Norovirus RNA fragment-containing pseudoviral particle and preparation method thereof

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Embodiment Construction

[0029] The embodiments of the present invention involve the following strains, reagents and instruments:

[0030] Escherichia coli MS2 phage (ATCC 15597B1) was purchased from ATCC, USA; pET-28b(+) plasmid / E. coli BL21(DE3) strain was purchased from Novagen, USA; Escherichia coli TGl strain was purchased from Stratagen; RNA extraction kit, plasmid Extraction and purification kit, DNA gel recovery kit, reverse transcriptase M-MLV are all products of Promega, USA; restriction endonuclease BamH Ⅰ. Nco Ⅰ and Hind Ⅲ Purchased from Dalian Bao Biological Engineering Company, T4 DNA Ligase purchased from Shanghai Shenggong Bioengineering Company, Taq DNA polymerase purchased from Beijing Tiangen Biochemical Technology Company; primers and oligonucleotide fragments of Norovirus GⅡ conserved region It was synthesized by Shanghai Shenggong Bioengineering Company, and the PCR amplification operation was performed using the American ABI company's PE7700 fluorescent quantitative PCR instrumen...

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Abstract

The invention discloses a norovirus RNA fragment-containing pseudoviral particle and a preparation method thereof. The pseudoviral particle is a spherical RNA-protein complex which is prepared by coating a norovirus RNA with an MS2 bacteriophage coat protein. The pseudoviral particle is prepared by the following steps: designing and manually synthesizing a primer and obtaining a target gene MS2 by a by PCR (polymerase chain reaction) method; connecting the target gene MS2 to a plasmid pET-28b(+) to obtain a recombinant plasmid pET-28b / MS2; connecting the recombinant plasmid pET-28b / MS2 with a NV fragment to obtain a recombinant plasmid pET-28b / MS2 / NV; guiding the obtained recombinant plasmid pET-28b / MS2 / NV into escherichia coli for prokaryotic expression; and precipitating a virus-like particle by a polyethylene glycol method, wherein the obtained virus-like particle is the norovirus RNA fragment-containing pseudoviral particle. The pseudoviral particle can be used as a standard product and a quality control product for RT-PCR detection, is non-infectious, safe, reliable, and good in stability, and has an RNase-resistant characteristic.

Description

technical field [0001] The invention relates to norovirus, in particular to a pseudovirion particle containing a norovirus RNA fragment and a preparation method thereof. Background technique [0002] Norovirus (Norovirus, NoV) was first detected by the American scholar Kapikin in 1972 using immunoelectron microscopy from the stool samples of gastroenteritis patients who broke out in a school in Norwalk, Ohio, USA in 1968. and named Norwalk-like virus (NLvS) after the place where it was discovered. In 1995, in the sixth classification report of the International Committee on Taxonomy of Viruses, Norovirus was officially included in the Caliciviridae and Norovirus genus. Diarrhea caused by norovirus has the characteristics of acute onset, rapid transmission, and wide coverage. Infection can occur throughout the year, often causing collective outbreaks. The infected targets are mainly school-age children and adults. Most of the outbreaks of this disease are seasonal, showing ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/04C12N15/40C12N15/63
Inventor 李智涛白永杰高秀菊董璠冯艳铭
Owner HENAN UNIV OF SCI & TECH
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