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Pseudovirus particle comprising hepatitis-A-virus nucleic acid fragment and preparation method of pseudovirus particle

A hepatitis A virus and pseudovirion technology, applied in the biological field, can solve the problems of inability to evaluate virus particle extraction and RNA reverse transcription detection steps, inability to evaluate virus particle extraction, affecting nucleic acid extraction, etc., and achieve non-infectious, Lower production cost and better stability

Inactive Publication Date: 2016-08-31
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

Based on references at home and abroad, it has been reported that inactivated virus is used as a positive standard sample, but there are residual inactivation reagents (such as formaldehyde, etc.) that affect nucleic acid extraction; some use plasmid DNA containing detection fragments as a positive standard sample, However, important detection steps such as virus particle extraction and RNA reverse transcription cannot be evaluated; some use in vitro transcribed RNA as a positive standard sample, and there are also problems that are easily degraded and cannot evaluate steps such as virus particle extraction

Method used

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  • Pseudovirus particle comprising hepatitis-A-virus nucleic acid fragment and preparation method of pseudovirus particle
  • Pseudovirus particle comprising hepatitis-A-virus nucleic acid fragment and preparation method of pseudovirus particle
  • Pseudovirus particle comprising hepatitis-A-virus nucleic acid fragment and preparation method of pseudovirus particle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of plasmid vector pET-QGBHAV

[0032] First, with reference to the Qbeta phage genome sequence (accession number is AB 971354) in the Genbank database and the HAV detection target sequence stipulated in the national standard GB / T 22287-2008, prepare the Qbeta phage maturation enzyme coding gene, DNA fragments including capsid protein coding genes, packaging site sequences, cDNA sequences corresponding to HAV nucleic acid fragments and auxiliary multiple cloning sites (hereinafter referred to as QGBHAV fragments) (see figure 1 ), the DNA fragment has the sequence shown in SEQ ID No.1 in the sequence listing. Auxiliary multiple cloning sites include, for example, Apa I, KpnI, Pst I, Spe I, Sph I, and Not I from the 5' end to the 3' end. The above DNA fragment was subcloned into the pUC-18 vector to obtain an intermediate plasmid vector, which was named pUC-QGBHAV.

[0033] Next, PCR amplification was performed using the intermediate plasmid vec...

Embodiment 2

[0045] Embodiment 2: the preparation of pseudovirus particle

[0046] Transform the plasmid vector pET-QGBHAV 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 culture 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, add ddH 2 O was adjusted to 100mL, autoclaved for later use.

[0047] 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 was adjusted to 100mL, autocl...

Embodiment 3

[0055] Embodiment 3: the detection of residual plasmid DNA in pseudovirion

[0056] Detect whether the pseudovirus particles prepared in Example 2 contain residual nucleic acid fragments of the plasmid pET-QGBHAV by PCR amplification method, design the upstream primer PpqGHAVF and the downstream primer PpqGHAVR, the sequences are as shown in Table 4, and the pair of primers can amplify the plasmid pET -A target region of about 134bp upstream and downstream of the hepatitis A virus target cDNA fragment contained in QGBHAV. The positive control was set up with the plasmid vector pET-QGBHAV as the template, and the ddH 2 O sets a negative control for the template. The reaction system is shown in Table 5.

[0057] Table 4

[0058] Primer

sequence

PPML

5'-CAGCATAAGCTTTTTCCGGAG-3'

PpqGHAVR

5'-GCGGCCGCTCTAGAGCAC-3'

[0059] table 5

[0060] Composition of the reaction system

Dosage

Template (pseudovirion stock solutio...

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Abstract

The invention provides a pseudovirus particle comprising a hepatitis-A-virus nucleic acid fragment and a preparation method of the pseudovirus particle. The pseudovirus particle comprises the hepatitis-A-virus nucleic acid fragment which can be used as a target for detecting hepatitis A virus. The preparation method includes creating a plasmid vector comprising a maturase coding gene, a capsid protein coding gene and a packaging site sequence of a coding Qbeta phage as well as a cDNA (complementary deoxyribonucleic acid) sequence corresponding to the hepatitis-A-virus nucleic acid fragment, performing transcription and / or translational expression on the plasmid vector in an escherichia coli cell, and performing separation and purification to obtain the pseudovirus particle. The pseudovirus particle can serve as a standard positive sample for detecting the hepatitis A virus, and is applied to hospitals, food sanitation testing organizations, quality testing organizations, scientific research institutions and other testing organizations. By the preparation method, the non-infectious pseudovirus particle with high concentration and stability can be prepared.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a pseudovirus particle containing hepatitis A virus nucleic acid fragments and a preparation method thereof. Background technique [0002] Hepatitis A virus (hepatitis A virus, HAV) is a non-enveloped single-stranded positive-sense RNA virus, belonging to the Hepadnavirus genus of the Picornaviridae family, and is the pathogen that causes the acute infectious disease of humans - hepatitis A. HAV is mainly transmitted through the fecal-oral route, and consumption of contaminated food or water can cause infection and even outbreaks. [0003] Contaminated clams and other bivalve mollusks are important carriers of hepatitis A transmission. Studies have shown that shellfish have a strong ability to enrich HAV, and can concentrate HAV in water by 10 to 1000 times. Consuming contaminated shellfish that has not been thoroughly cooked can lead to serious public health concerns. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/70C12R1/19
CPCC12N7/00C12N15/70C12N2770/32431C12N2800/101
Inventor 姚琳江艳华李风铃朱文嘉郭莹莹庞倩倩王联珠翟毓秀
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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