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Tick-borne forest encephalitis virus armored RNA standard substance and preparation method thereof

A forest encephalitis virus and standard substance technology, which is applied in the field of forest encephalitis virus armored RNA standard material and its preparation, can solve the problem that the forest encephalitis virus armored RNA standard material is not disclosed, and achieve the effect of being resistant to RNase enzyme degradation

Pending Publication Date: 2021-10-08
NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no relevant research reports on the armored RNA standard substance of forest encephalitis virus and its preparation method at home and abroad

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Obtaining and Identification of Armored RNA of Gene C of Forest Encephalitis Virus

[0025] 1. Phage MS2 CP gene amplification

[0026] Design and synthesize fragments CPP1 and CPP2 containing phage MS2 CP protein sequence (sequences such as SEQ ID NO: 1, 2), and insert Kpn I and BamHI restriction sites at both ends of the sequence respectively, and use the phage MS2 plasmid kept in the laboratory The pUC18-MS2 sequence was used as a template, and the MS2 phage gene sequence CP was amplified by PCR using CPP1 and CPP2. PCR reaction system: 10×PCR buffer 2µL, 2.5mM dNTP 1.6µL each, 5U / µL rTaq enzyme 0.4µL, MgCl 2 1.2µL, 1µL of 20pmol / L primer CP P1, 1µL of 20pmol / L primer CP P2, 11.8µL of double distilled water, 1µL of plasmid pUC18-MS2. Reaction conditions: 94°C for 5min, 94°C for 30sec, 52°C for 30sec, 72°C for 1min, after 35 cycles, 72°C for 7min. Use 1.5% agarose gel electrophoresis to observe the effect of PCR amplification, and observe whether there is a target...

Embodiment 2

[0037] Identification of the armored RNA of forest encephalitis virus

[0038] Take 20 µL of forest encephalitis virus armored RNA and use 100U RNase A and 2U DNaseI to act at 37°C for 1 hour, use QiaGen RNeasy Mini Kit extraction kit (purchased from Qiagen) to extract RNA, and use forest encephalitis virus fluorescent quantitative PCR primers to amplify, Reaction system: 2 × One Step RT-PCR Buffer 12.5μL, TaKaRa EX TaqHS 0.5μL, PrimeScript RT Enzyme Mix 0.5μL, RNase Free dH 2 O 7.5 μL, TBEV-F 0.5 μL, 20 μmol / L TBEV-R 0.5 μL, 50 μmol / L TBEV-P 0.3 μL (sequence such as SEQ ID NO: 5, 6, 7), template 2 μL. Reaction conditions: 45°C for 10 minutes, 95°C for 15 minutes, then press 95°C for 5s, 60°C for 30s, cycle 45 times, single-channel fluorescence detection at 60°C, observe the fluorescence quantitative PCR amplification curve, the Ct value of the armored RNA sample is lower than 30 and There is a standard amplification curve.

Embodiment 3

[0040] Armored RNA Quality Control Stability Verification

[0041] 1. Storage stability experiment under different environmental conditions

[0042] Use RNase A and DNaseI to digest the crude virus-like particle product completely, place it at 37°C, 4°C, -20°C for 10d, 20d, 30d, 40d, 50d, and store the sample at -80°C as a control , RNA was extracted using QiaGen RNeasy MiniKit, and detection results were amplified using fluorescent quantitative PCR primers for forest encephalitis virus. The results show that virus-like particles can be stored at 37°C for 10 days, and can be stored at 4°C for 30 days, with a Ct value lower than 40 and an obvious amplification curve; stored at -20°C for 50 days, the Ct value is lower than 40 and has obvious amplification curve.

[0043] 2. Different quality control enzyme digestion experiments

[0044] The positive quality control of the forest encephalitis virus vaccine preserved in the laboratory and the armored RNA of the prepared forest ...

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Abstract

The invention discloses a tick-borne forest encephalitis virus armored RNA standard substance and a preparation method thereof. The tick-borne forest encephalitis virus armored RNA standard substance is characterized in that an expression vector of armored RNA is pET32a-CP-C plasmid, the plasmid comprises a phage MS2 CP protein sequence fragment and a tick-borne forest encephalitis virus C protein sequence fragment; the preparation method comprises the step of phage MS2 CP gene amplification; constructing the expression vector pET32a-CP, synthesizing the tick-borne forest encephalitis virus C protein sequence, and constructing an expression vector pET32a-CP-C; and finally, transferring the tick-borne forest encephalitis virus armored RNA expression vector pET32a-CP-C into an escherichia coli prokaryotic expression strain BL21, collecting thalli through induced expression centrifugation, conducting centrifugation after ultrasonic disruption, taking supernatant, and obtaining the tick-borne forest encephalitis virus armored RNA standard substance. The tick-borne forest encephalitis virus armored RNA standard substance has the advantages of being stable, safe and resistant to ribonucleic acid.

Description

technical field [0001] The invention relates to an armored RNA standard substance, in particular to an armored RNA standard substance of forest encephalitis virus and a preparation method thereof. Background technique [0002] According to the report of the World Health Organization, the annual incidence of global forest encephalitis is about 20,000 cases. In recent years, the number of cases of forest encephalitis caused by tick bites in my country has also increased significantly. Tick-borne Forest Encephalitis virus (TBEV) belongs to the Flavivirus genus (Flavivirus) of the Flaviviridae family. The main carrier of forest encephalitis virus is ticks, and the source of infection is wild rodents and hedgehogs. After ticks feed on the blood of infected animals, the virus reproduces in the ticks, and after being bitten by virus-carrying ticks, humans and other animals can become infected. my country's Inner Mongolia, Heilongjiang, and Jilin are the main epidemic areas of fo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70C12R1/19
CPCC07K14/005C12N15/70C12N2770/24122C12N2795/00022C12N2795/00023C07K2319/00
Inventor 马思杰胡群童淑梅邹春颖
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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