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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: 2012-07-11
HENAN UNIV OF SCI & TECH
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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] Embodiments of the present invention relate to following strains, reagents and instruments:

[0030] Escherichia coli MS2 phage (ATCC 15597B1) was purchased from ATCC Company of the United States; pET-28b (+) plasmid / Escherichia coli BL21 (DE3) bacterial strain was purchased from American Novagen Company; Escherichia coli TG1 bacterial strain was purchased from Stratagen Company; Extraction and purification kit, DNA gel recovery kit, and reverse transcriptase M-MLV are all products of Promega Company in the United States; restriction endonuclease Bam H I. Nco I and Hind Ⅲ was purchased from Dalian Bao Biological Engineering Company, T4 DNA ligase was purchased from Shanghai Sangon Bioengineering Company, Taq DNA polymerase was purchased from Beijing Tiangen Biochemical Technology Company; It was synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the PCR amplification operation used the PE7700 fluorescent quantitative PCR instrument of ABI Company of the U...

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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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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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