Pseudoviral particle containing hepatitis e virus RNA (Ribose Nucleic Acid) fragment and preparation method thereof

A technology of hepatitis E virus and pseudovirus particles, which is applied in the field of pseudovirus particles containing RNA fragments of hepatitis E virus and its preparation, which can solve the problem of failure to monitor the whole process of nucleic acid extraction and reverse transcription, and the existence of biological safety hazards , poor stability and other problems, to achieve the effect of solving stability problems, not easy to degrade, and good stability

Inactive Publication Date: 2014-07-16
JINZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology solves many disadvantages of traditional RNA quality control products (either poor stability, or potential biological safety hazards, or failure to achieve the purpose of monitoring the whole process of nucleic acid extraction and reverse transcription, etc.)

Method used

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  • Pseudoviral particle containing hepatitis e virus RNA (Ribose Nucleic Acid) fragment and preparation method thereof
  • Pseudoviral particle containing hepatitis e virus RNA (Ribose Nucleic Acid) fragment and preparation method thereof
  • Pseudoviral particle containing hepatitis e virus RNA (Ribose Nucleic Acid) fragment and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The construction of the pseudoviral vector pET-28b / MS2 before embodiment 1 HEV

[0059] 1. Artificially synthesize the MS2 phage gene sequence (NC_001417) in the GenBank database, and design the primer pair Pri-MS2F and Pri-MS2R of the assembly protein and envelope protein gene through the synthetic sequence, and artificially synthesize it.

[0060] Pri-MS2F: 5'-GGT CCATGG CCTTTCGGGGTCCTGCTCAACTT-3' (the underline indicates that the introduced restriction site is Nco I)

[0061] Pri-MS2R: 5'-GGT GGATCC GCTGAGGGAATCGGGTTTCCATCTT-3' (the underline indicates that the restriction site introduced is BamH I)

[0062] 2. PCR amplification, the PCR product was connected to the cloning T vector pMD18-T, and the pMD18-T / MS2 plasmid was constructed.

[0063] 3. The expression vectors of recombinant plasmids pMD18-T / MS2 and pET28b(+) were double-digested with fast enzymes Nco I and BamH I respectively (both are preserved in our laboratory). The enzyme digestion system is as f...

Embodiment 2

[0070] The construction of embodiment 2 HEV pseudovirus vector pET-28b / MS2 / HEV

[0071] 1. Referring to the HEV (DQ279091.2) gene type IV ORF2 conserved region sequence, directly synthesize the HEV ORF2 overlapping ORF3 conserved region sequence. According to the above-mentioned synthetic gene sequence, the primer pairs Pri-ORF2 / 3F and Pri-ORF2 / 3R were designed, so that the fragment tails contained 24 artificially introduced Poly-A tails.

[0072] Pri-ORF2 / 3F: 5'- GGATCC CCTATGCTGCCCGCGCCACCG-3' (the underline indicates that the introduced enzyme cutting site is BamH I)

[0073] Pri-ORF2 / 3R: 5'- AAGCTT TTTTTTTTTTTTTTTTTTTTTTTTACGGCGAAGCCCCAGCT-3' (the underline means that the introduced enzyme cutting site is HindIII).

[0074] 2. PCR amplification, the PCR product was connected to the cloning T vector pMD18-T, and the DMD18-T / ORF2 / 3 plasmid was constructed.

[0075] 3. Double-digest recombinant plasmid pMD18-T / ORF2 / 3 and HEV propseudoviral vector pET-28b / MS2 with fast e...

Embodiment 3

[0082] Embodiment 3 Preparation of HEV pseudovirus pET-28b / MS2 / HEV.

[0083] 1. Induce and express 100mL of recombinant bacteria DH5α / pET-28b / MS2 / HEV with an OD600 value of about 0.5-1.0, with a final concentration of IPTG of 0.5mol / L, 37°C, 200r / m, 4h; centrifuge at 12000r / m, After 5 minutes, the supernatant was discarded, and the cell pellet was collected.

[0084] 2. Resuspend the bacterial pellet in 10mL lysate (50mM Tris-HCl, pH8.5-9.0, 2mM EDTA, 100mM NaCl, 0.5% Triton X-100, lysozyme 10mg / L), and act in a water bath at 37°C for 30min. Sonicate at 400W at 400W, 5s, 10s interval, 5min in total, and centrifuge at 10,000r / m for 10min at 4°C after the bacterial liquid is transparent, and collect the supernatant. Add ribonuclease and deoxyribonuclease I to a final concentration of 1 mg / L, act in a water bath at 37°C for 1 hour, centrifuge at 10,000 r / m for 10 minutes at 4°C, and collect the supernatant. Add NaCl to a final concentration of 0.5mol / L, add an equal volume of 1...

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Abstract

The invention provides a pseudoviral particle containing a hepatitis e virus (HEV) RNA (Ribose Nucleic Acid) fragment and a preparation method thereof. The pseudoviral particle is an RNA-protein complex formed by coating hepatitis e virus RNA by an MS2 bacteriophage coat protein, and the RNA-protein complex is spherical. The preparation method comprises the steps of designing and artificially synthesizing a primer to obtain a target gene MS2 through a PCR (Polymerase Chain Reaction) method, connecting the target gene MS2 to a plasmid pET (polyethylene glycol terephthalate)-28b(+) to obtain a recombinant plasmid pET-28b/MS2/HEV, guiding the recombinant plasmid into escherichia coli for prokaryotic expression, and settling a virus-like particle by adopting a polyethylene glycol method, wherein the virus-like particle is the pseudoviral particle containing the hepatitis e virus RNA fragment. The pseudoviral particle provided by the invention can be used as a standard substance and a quality control product of general HEV gene I-IV type RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection, and has the characteristics no infectivity, safety, reliability, high stability and resistance to ribonuclease.

Description

technical field [0001] The invention relates to hepatitis E virus, in particular to a pseudovirion particle containing hepatitis E virus RNA fragment and a preparation method thereof. Background technique [0002] Hepatitis E virus (HEV) is a hepatitis virus transmitted through the digestive tract and blood. The infection spectrum of the virus is very wide, and the currently confirmed infection hosts include not only humans, but also other animals, such as pigs, monkeys, and chickens. There are four genotypes of HEV, type I and type IV are limited to human-to-human transmission, type III and type IV are cross-transmission between humans and animals; clinical patients with human infection are mostly mild to moderate hepatitis, often self-limiting, but pregnant women suffer from type E The serious case fatality rate of hepatitis can reach 20%. Therefore, the World Health Organization (WHO) identified HEV as a newly discovered zoonotic pathogen in this century, and the global ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/04C12N15/70C12N15/66C12R1/93
Inventor 高慎阳查恩辉周铁忠李丹丹董筱萍
Owner JINZHOU MEDICAL UNIV
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