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Preparing method for GI type norovirus virus-like particles and application thereof

A technology of virus-like particles, applied in the preparation and application of GI-type norovirus virus-like particles, can solve the problems of biological safety hazards, easy to be degraded by ribonuclease, unable to reflect the influence of nucleic acid extraction process, etc., to achieve a solution The problem of stability, the convenience of large-scale promotion and application, and the effect of avoiding the risk of false negatives

Inactive Publication Date: 2016-04-20
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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  • Preparing method for GI type norovirus virus-like particles and application thereof
  • Preparing method for GI type norovirus virus-like particles and application thereof
  • Preparing method for GI type norovirus virus-like particles and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of NV-GI-VLPs

[0037] The preparation method of the virus-like particle standard substance containing NV-GI gene, comprises the following steps:

[0038] (1) Construction of VLPs recombinant plasmid pET-NH-MS2his containing histidine tag

[0039] 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.

[0040] 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, an...

Embodiment 2

[0048] The characteristic detection of embodiment 2NV-GI-VLPs

[0049] For the characteristic detection of the virus-like particle standard substance containing the NV-GI gene, the steps are as follows:

[0050] (1) RNA extraction and RT-PCR detection in NV-GI-VLPs

[0051] Use 100 μL of the purified virus-like particle solution to extract RNA as a template, and perform RT-PCR amplification according to the NV-GI primer and probe sequence. 1 μL, RNA template 5 μL, ddH2O make up 25 μL, the reaction conditions were reverse transcription at 50°C for 30 minutes, extinguishing and denaturing at 95°C for 10 minutes, then 95°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute, 40 cycles, and finally 72°C for 10 minutes. The amplified product was electrophoresed on 1.0% agarose gel to detect about 86bp, and sent for sequencing. The electrophoresis picture is as follows figure 2 .

[0052] (2) Detection of NV-GI-VLPs resistance to RNaseA

[0053] Take 2 parts of 50 μL purified ex...

Embodiment 3

[0060] Norovirus RNA Detection Application in Example 3 Strawberry Berries

[0061] Using NV-GI-VLPs as a quality control sample to detect Norovirus RNA in strawberry berries, the steps are as follows:

[0062] (1) Enrichment of viruses in fruits, vegetables and berries

[0063] a) 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 100 μL of coliphage MS2 standard sample at the same time.

[0064] b) Incubate at room temperature with constant shaking at 60 rpm for 20 min.

[0065] 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.

[0066] c) 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 super...

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Abstract

The invention discloses a preparing method for virus-like particles containing norovirus GI genes. The virus-like particles are an RNA-protein complex formed by wrapping NV-GI RNA with MS2 bacteriophage encoding coat protein; a primer is designed and artificially synthesized, the norovirus GI gene sequence is amplified by means of the RT-RCR technology and cloned into pNH-MS2his, and pronucleus recombinant expression plasmid pNH-MS2his-NV-GI is constructed. The obtained pNH-MS2his-NV-GI plasmid is converted in expression escherichia coli BL21(DE3) to carry out inducible expression. The virus-like particles can serve as standards and quality control products for detecting RT-PCR, are free of infectivity, safe, reliable and good in stability, and have the advantage of resisting ribonuclease.

Description

technical field [0001] The invention relates to a preparation method and application of virus-like particles containing Norovirus GI 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 responsibl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/11C12N15/70C12N7/04C12Q1/70C12Q1/68C12R1/93
CPCC07K14/005C12N7/00C12N15/70C12N2770/16022C12N2770/16023C12N2800/101C12Q1/701C12Q2600/166
Inventor 张晓文王群郑小龙赵玉然岳志芹房保海梁成珠贾俊涛王宫璞
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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