Preparation method for GII Norovirus virus-like particles and application thereof

A NV-GII, plasmid technology, applied in biochemical equipment and methods, viruses, viruses/phages, etc., can solve the problems of not reflecting the impact of nucleic acid extraction process, hidden dangers of biological safety, easy to be degraded by ribonuclease, etc., to achieve convenient The effect of large-scale promotion and application, solving stability, and avoiding the risk of false negatives

Inactive Publication Date: 2016-04-06
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus culture or cDNA obtained by reverse transcription of RNA is usually used as a standard, but the former has hidden dangers in biological safety and is easily degraded by ribonuclease, while the latter cannot reflect the influence of nucleic acid extraction process and reverse transcription on the test

Method used

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  • Preparation method for GII Norovirus virus-like particles and application thereof
  • Preparation method for GII Norovirus virus-like particles and application thereof
  • Preparation method for GII Norovirus virus-like particles and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of NV-GII-VLPs

[0039] The preparation method of the virus-like particle standard substance containing NV-GII gene, comprises the following steps:

[0040] (1) Construction of VLPs recombinant plasmid pET-NH-MS2his containing histidine tag

[0041] The pET32a plasmid was digested with XbaI and NcoI, and the sequence between the two restriction sites of XbaI and NcoI was removed, and the artificially synthesized 5'-PO4-CTAGATTACAAGGC-3' and 5'-PO4-CATGGCCTTGTAAT-3' were Complementary oligonucleotides were inserted into it after renaturation, and the recombinant plasmid pET-NH was constructed and identified by sequencing.

[0042] A one-step amplification kit was used to amplify the MS2 target fragment with MS2Pf1 and MS2Pr1 as primers and MS2RNA as a template. The amplified product was detected by agarose gel electrophoresis, and the target fragment was recovered with an agarose gel electrophoresis recovery kit (TaKaRa), eluted with 50 μL ddH2O, ...

Embodiment 2

[0050] The characteristic detection of embodiment 2NV-GII-VLPs

[0051] For the characteristic detection of the virus-like particle standard substance containing the NV-GII gene, the steps are as follows:

[0052] (1) RNA extraction and RT-PCR detection in NV-GII-VLPs

[0053] Use 100 μL of the purified virus-like particle solution to extract RNA as a template, and perform RT-PCR amplification with the NV-GII primer and probe sequence in Power 4. The RT-PCR system is: 5×RT-PCR buffer 5 μL, dNTPmix 1 μL, EnzymeMix 1 μL, primer NF 1 μL each of NR and NR, 5 μL of RNA template, 25 μL of ddH2O supplementation, the reaction conditions are: reverse transcription at 50°C for 30 minutes, denaturation at 95°C for 10 minutes, then 1 minute at 95°C, 1 minute at 55°C, 1 minute at 72°C, 40 cycles, and finally 72°C Extend for 10 minutes. The amplified product was detected by 1.0% agarose gel electrophoresis, and a band of about 89bp could be seen ( figure 2 ), sent for sequencing.

[00...

Embodiment 3

[0062] Norovirus RNA Detection Application in Example 3 Strawberry Berries

[0063] Using NV-GII-VLPs as a quality control sample to detect Norovirus RNA in strawberry berries, the steps are as follows:

[0064] (1) Enrichment of viruses in fruits, vegetables and berries

[0065] Take 5-10 strawberries or 25g test sample and add it to the bagpage; add 35mL TGBE buffer solution and 30U pectinase Aspergillus niger, and add 100μL of coliphage MS2 standard sample at the same time.

[0066] a) Incubate at room temperature with constant shaking at 60 rpm for 20 min.

[0067] Note: For acidic soft fruits, the pH of the eluate should be monitored at 10-min intervals during the incubation. If the pH is below 9.0, adjust to 9.5 with NaOH solution. If the pH value is adjusted, the incubation time should be extended by 10 min.

[0068]b) Collect the eluate in a 50 mL sterile centrifuge tube, centrifuge at 10,000×g for 30 min, and centrifuge at 4° C. to clarify. Transfer the supernata...

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Abstract

The invention discloses a preparation method for virus-like particles containing Norovirus GII genes. The virus-like particles are formed in the way that MS2 phage coding coat protein is wrapped with RNA-protein complexes containing NV-GII RNA; a coding histidine tag sequence is inserted in a beta hairpin loop structure sequence of the MS2 phage coding coat protein to construct MS2 phage coat protein containing recombined histidine tags and pNH-MS2his recombinant plasmids expressing mature zymoprotein genes. The Norovirus GII genes are amplified through the RT-RCR technology and cloned into pNH-MS2his to construct pronucleus recombined expression plasmids pNH-MS2his-NV-GII. The obtained pNH-MS2his-NV-GII plasmids are transformed into expression escherichia coli BL21 (DE3) for inducible expression. A purification column of Ni-NTA is adopted to purify the virus-like particles, and the obtained virus-like particles contain the Norovirus GII genes and are named NV-GII-VLPs. The NV-GII virus-like particles can serve as standard substances and quality control substances for RT-PCR detection, are free of infectivity, safe, reliable and good in stability and have the advantage of ribonuclease resistance.

Description

technical field [0001] The invention relates to a preparation method and application of virus-like particles containing Norovirus GII gene. Background technique [0002] Norovirus, also known as Norwalk Virus (Norwalk Viruses, NV), is the prototype representative strain of the Norovirus (Norovirus, NV) genus in the Human Calicivirus (HuCV) family. It is a group of virus particles with similar morphology and slightly different antigenicity. Norovirus was first isolated from the feces of patients with acute diarrhea in Norwalk, USA in 1968. In August 2002, the Eighth International Virus Nomenclature Committee approved the name Norovirus, which together with the Sapporo-like virus found in Japan, was collectively called Human Calicivirus. Norovirus infectious diarrhea is prevalent all over the world, and infection can occur throughout the year. The infected objects are mainly adults and school-age children, and the incidence is high in cold seasons. Noroviruses are responsib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/70C12N7/04C12Q1/70C12Q1/68
CPCC12N7/00C12N15/70C12N2770/16023C12Q1/701C12Q2600/166
Inventor 梁成珠王群孙涛徐文菲张晓文岳志芹房保海赵玉然贾俊涛王宫璞
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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