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

A pseudovirion and plasmid vector technology, applied in the field of high-stability pseudovirions and plasmid vectors used for their preparation, can solve problems such as hidden dangers of biological safety, evaluation, and uneven virus content, and reduce production costs and copy numbers. High and stable effect

Active Publication Date: 2016-09-21
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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...

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of plasmid vector pET-QINVGII

[0037]First, refer to the Qbeta phage genome sequence (accession number is AB971354) in the Genbank database and the GII type norovirus detection target sequence specified in ISO / T15216-2 2012, and prepare the coding gene comprising the Qbeta phage maturation enzyme by, for example, artificial synthesis. , the capsid protein coding gene, the packaging site sequence, the cDNA sequence corresponding to the GII type norovirus nucleic acid fragment and the DNA fragment including the auxiliary multiple cloning site (hereinafter referred to as the QINVGII fragment) (see figure 1 ), the specific sequence of the DNA fragment is shown in SEQ ID No.1 in the sequence listing.

[0038] Auxiliary multiple cloning sites include, for example, Apa I, Kpn I, Pst I, Spe I, Sph I, and Not I from the 5' end to the 3' end. The above nucleic acid fragment was subcloned into the pUC-18 vector to obtain an intermediate plasmid vector ...

Embodiment 2

[0049] Embodiment 2: the preparation of pseudovirus particle

[0050] Transform the plasmid vector pET-QINVGII prepared in Example 1 into, for example, Escherichia coli BL21 competent cells, and then spread it on a nutrient agar plate containing kanamycin (final concentration: 50 μg / mL), and incubate at 37°C for 18h . The specific preparation method of nutrient agar plate is as follows: Tryptone 1g, NaCl 0.5g, yeast extract 0.5g, agar powder 1.5g, dissolved in 90mL ddH 2 In O, adjust the pH value to 7.0-7.2, and then add ddH 2 O and dilute to 100mL, autoclaved for later use.

[0051] Then, a single colony spot was picked from the cultured plate above, and inoculated in 3 mL of liquid LB medium containing kanamycin (final concentration: 50 μg / mL), and cultured at 37° C. for 18 h. The specific preparation method of liquid LB medium is: tryptone 1g, NaCl 0.5g, yeast extract 0.5g, dissolved in 90mL ddH 2 In O, adjust the pH value to 7.0-7.2, add ddH 2 O and dilute to 100mL,...

Embodiment 3

[0059] Embodiment 3: the detection of residual plasmid DNA in pseudovirion

[0060] Detect whether the pseudovirus particles prepared in Example 2 contain the nucleic acid of residual plasmid pET-QINVGII by PCR amplification method, design upstream primer PpqinvF and downstream primer PpqinvR, the sequences are as shown in Table 4, and the pair of primers can amplify the plasmid pET - A total of 150 bp target region upstream and downstream of the GII type norovirus target cDNA fragment contained in QINVGII. Set the positive control with the plasmid vector pET-QINVGII as the template, and ddH 2 O sets a negative control for the template. The reaction system is shown in Table 5.

[0061] Table 4

[0062] Primer

sequence

PPML

5'-GACAGCATAAGCTTTTTCC-3'

QUR

5'-GCGGCCGCTCTAGAGCAC-3'

[0063] table 5

[0064] Composition of the reaction system

Dosage

Template (pseudovirion stock solution)

1.0 μL

PpqinvF (...

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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...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/70C12R1/19
CPCC12N7/00C12N15/70C12N2770/16021C12N2800/101
Inventor 姚琳庞倩倩江艳华李风铃朱文嘉郭莹莹王联珠翟毓秀
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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