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Armored RNA standard substance containing Seoul virus G2 protein gene and preparation method of armored RNA standard substance

A Seoul virus and standard substance technology, applied in the biological field, can solve the problems that DNA templates cannot participate in the nucleic acid extraction process and reverse transcription process, and cannot be detected in the whole process monitoring, etc., achieving no biological safety hazards, easy storage, and RNase resistance. The effect of enzymatic degradation

Pending Publication Date: 2020-08-25
中华人民共和国大榭海关 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since virus preparation needs to be carried out in a high-standard biosafety laboratory, and due to restrictions on transportation and storage, it is used as a quality control standard substance
Various hemorrhagic fever virus detection kits currently on the market in China generally use synthetic RNA or DNA. RNA templates are easily degraded by RNase enzymes in the environment, and DNA templates cannot participate in the nucleic acid extraction process and reverse transcription process, and cannot be used for the entire detection process. monitor

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Obtaining and identifying armored RNA of the G2 protein gene of Seoul virus virus.

[0027] (1) Phage MS2 CP gene amplification

[0028]The primers CPP1 and CPP2 containing the bacteriophage MS2 CP protein sequence fragment were designed and synthesized, and their sequences were shown in SEQ ID NO:1 and SEQ ID NO:2, and Kpn I and BamHI restriction sites were inserted at both ends of the sequence respectively. Using the phage MS2 plasmid pUC18-MS2 sequence kept in the laboratory as a template, the MS2 phage gene sequence MS2 CP was amplified by PCR using CPP1 and CPP2. PCR reaction system: 10X PCR Buffer 2μL, dNTP (2.5mM each) 1.6μL, rTaq (5U / μL) 0.4μL, Mgcl2 1.2μL, primer CP P1 / CP P2 (20pmol / L) 1μL, DDW 11.8μL, plasmid pUC18 - MS2 1 μL. Reaction conditions: 94°C for 5 min, 94°C for 30 sec, 52°C for 30 sec, 72°C for 1 min (35 cycle), and 72°C for 7 min. Use 1.5% agarose gel electrophoresis to observe the effect of PCR amplification, and observe whether ther...

Embodiment 2

[0035] Example 2: Identification of Seoul virus armored RNA.

[0036] Take 20 μL of Seoul virus, use 100U RNase A and 2U DNaseI, and act at 37°C for 1h, use the QiaGenRNeasy Mini Kit extraction kit (purchased from Qiagen) to extract RNA, and use Seoul virus universal primers for RT-PCR and PCR amplification respectively, RT- PCR cDNA synthesis reaction system: RNA 10μL, HTP1 (20pmol / L) 2μL (its sequence is shown in SEQ ID NO: 5), 10X dNTP (10pmol / L) 1μL, RNasin (40U / μL) 0.5μL, AMV reversal Transcriptase (10U / μl) 1 μL, 5X AMV reverse transcriptase buffer 4 μL, DEPC water 1.5 μL. Reaction conditions: 1h at 42°C, 10min at 72°C. DNA amplification: 10X PCR Buffer 2μL, dNTP (2.5mM each) 1.6μL, rTaq (5U / μL) 0.4μL, Mgcl2 1.2μL, primer HT P2 / HT P3 (20pmol / L) 1μL (the sequence is as shown in SEQ ID NO :6 and SEQ ID NO:7), DDW 11.8 μL, cDNA 1 μL. The reaction conditions were 94°C for 5min, 94°C for 30s, 56°C for 30sec, 72°C for 1min (35cycle), and 72°C for 10min. The PCR amplification...

Embodiment 3

[0037] Example 3: Stability verification of armored RNA quality control products.

[0038] 1. Storage stability experiment under different environmental conditions

[0039] Use RNase A and DNaseI to digest the crude virus-like particle product completely, place it at 37°C, 4°C, -20°C for 15d, 30d, 45d, 60d, 75d, 90d, and store the sample at -70°C As a control, QiaGen RNeasyMini Kit was used to extract RNA, amplified with Seoul virus universal primers, and then agarose gel electrophoresis was used to detect the experimental results. The results of electrophoresis showed that the virus-like particles could be stored at 37°C for 15 days, and the electrophoretic bands could still be observed after 30 days at 4°C, and the electrophoretic bands could be observed at -20°C for 75 days.

[0040] 2. Different quality control enzyme digestion experiments

[0041] The positive quality control of Seoul virus RNA, Seoul virus vaccine, and prepared Seoul virus armored RNA preserved in the ...

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Abstract

The present invention discloses an armored RNA standard substance containing a Seoul virus G2 protein gene and a preparation method of the armored RNA standard substance. The RNA standard substance uses a phage MS2 capsid protein to resist degradation by ribonuclease in the external environment and protect internal RNA fragments. A prokaryotic expression technology is used to express the MS2 phagecapsid protein-packaged Seoul virus part fragment gene, and the armored RNA standard substance is stable to prepare, bases Seoul virus G2 envelope protein part fragment sequence as a target and is suitable for quality control of a Seoul virus nucleic acid detection method. The prepared Seoul virus armored RNA standard substance has characteristics of stability, no biological safety hazard, resistance to RNase enzyme degradation, etc., is convenient for storage, transportation and use, can perform quality control on the whole process of virus extraction and RT-PCR of Seoul virus detection, andis simple in operation, safe and effective

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a standard substance containing Seoul virus G2 protein gene armored RNA and a preparation method. Background technique [0002] In recent years, human infection with hemorrhagic fever with renal syndrome caused by Seoul virus has often been reported and has become an important public health problem; Seoul virus belongs to the genus Hantaan virus of the Bunyaviridae family. . The main host of Seoul virus is the Rattus norvegicus. In my country, the black house rat, yellow-breasted rat and Mus musculus can all carry Seoul virus. my country is the country most seriously affected by hantavirus, and the number of hemorrhagic fever with renal syndrome accounts for more than 90% of the reported cases in the world. In 1981, an outbreak of hemorrhagic fever caused by Seoul virus broke out in the border area between Henan and Shanxi in China. Moreover, Seoul virus is the only ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/40C12N15/66C07K14/175
CPCC12N15/70C12N15/66C07K14/005C12N2760/12122C12N2760/12123C12N2830/60
Inventor 马思杰胡群童淑梅邹春颖谢东华
Owner 中华人民共和国大榭海关
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