Preparing method for HAV virus-like particle and application thereof

A virus-like, particle-like technology, applied to biochemical equipment and methods, positive single-stranded RNA viruses, viruses, etc., can solve problems such as easy degradation by ribonucleases, inability to reflect the impact of nucleic acid extraction process, and biological safety hazards, etc., to achieve It is convenient for large-scale promotion and application, avoids the risk of false negatives, and solves the effect of stability problems

Inactive Publication Date: 2016-04-20
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • 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 HAV virus-like particle and application thereof
  • Preparing method for HAV virus-like particle and application thereof
  • Preparing method for HAV virus-like particle and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 prepares HAV5'-UTR-VLPs

[0038] The preparation method of the virus-like particle standard substance containing HAV5'-UTR gene, comprises the following steps:

[0039] (1) Construction of VLPs recombinant plasmid pET-NH-MS2his containing histidine tag

[0040] 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.

[0041] 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 ddH2...

Embodiment 2

[0049] The characteristic detection of embodiment 2HAV5'-UTR-VLPs

[0050] The characteristic detection of the standard substance of virus-like particles containing the HAV-UTR gene, the steps are as follows:

[0051] (1) RNA extraction and RT-PCR detection in HAV5'-UTR-VLPs

[0052] Use 100 μL of the purified virus-like particle solution to extract RNA as a template, and perform RT-PCR amplification according to the primer and probe sequences of HAV5'-UTR in Power 4. The RT-PCR system is: 5×RT-PCR buffer 5 μL, dNTPmix 1 μL, EnzymeMix 1 μL, Primers HAV-F and HAV-R each 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 1 minute at 95°C, 1 minute at 55°C, 1 minute at 72°C, and 40°C. cycle, and finally extended at 72°C for 10 min; the amplified product was detected by 1.0% agarose gel electrophoresis to 173bp, see figure 2 , send for sequencing.

[...

example 3

[0061]Example 3. Application of Hepatitis A Virus RNA Detection in Strawberry Berries

[0062] HAV5'-UTR-VLPs were used as quality control samples to detect hepatitis A virus RNA in strawberry berries, and the steps were as follows.

[0063] (1) Enrichment of viruses in strawberry berries

[0064] 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.

[0065] b) Incubate at room temperature with constant shaking at 60 rpm for 20 min.

[0066] 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.

[0067] 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. ...

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Abstract

The invention discloses a preparing method for a virus-like particle containing a hepatitis A virus (HAV)5'-UTR gene. The virus-like particle is an RNA-protein complex formed by wrapping HAV5'-UTR RNA with MS2 bacteriophage encoding coat protein; the encoding histidine label sequence is inserted into a beta hairpin loop structure sequence of the MS2 bacteriophage encoding coat protein, and the MS2 bacteriophage encoding coat protein containing a recombined histidine label and pNH-MS2his recombinant plasmid expressing a maturase protein gene are constructed. The RT-RCR is used for amplifying the HAV5'-UTR gene, the HAV5'-UTR gene is cloned into pNH-MS2his, and pronucleus recombinant expression plasmid is constructed and named pNH-MS2his-HAV5'-UTR. The obtained pNH-MS2his-HAV5'-UTR plasmid is converted into expression escherichia coli BL21 for inducible expression. A purification column of Ni-NTA is adopted for purifying the virus-like particle. The obtained virus-like particle is the virus-like particle containing the HAV5'-UTR gene and is named HAV5'-UTR-VLPs. The HAV5'-UTR-VLPs can serve as standards and quality control products for detecting RT-PCR, is free of infectivity, safe, reliable and good in stability, and has the advantage of resisting ribonuclease.

Description

technical field [0001] The invention relates to a preparation method and application of virus-like particles containing hepatitis A virus HAV5'-UTR gene. Background technique [0002] In 1973, Feinslone first discovered hepatitis A virus (Hapatitis Avirus, HAV) in the feces of patients in the acute phase by immunoelectron microscopy. It belongs to Picornaviridae, a new type of enterovirus 72, and is a common food-borne disease virus. HAV has strong resistance to the outside world, acid and alkali resistance, and can survive for 1 week at room temperature, and can survive in dry feces at 25°C 30 days. After human infection with HAV, most of them manifested as subclinical or recessive infection, and a few manifested as acute hepatitis A, seriously endangering the health of the people. Hepatitis A virus is distributed all over the world, and my country, as one of the high-incidence areas, has a very high incidence rate. Hepatitis A virus is mainly transmitted through food. W...

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
CPCC12N7/00C12N15/70C12N2770/32423C12Q1/706C12Q2600/166Y02A50/30
Inventor 张晓文房保海孙明君尹伟力岳志芹王群梁成珠王宫璞贾俊涛
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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