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A kind of dna molecule comprising the whole genome cdna of chikungunya virus and its use

A technology of chikungunya virus and DNA molecules, which is applied in the field of DNA molecules, can solve the problems of no effective therapeutic drugs and preventive vaccines, and achieve the effects of improving efficiency, shortening the construction process, and avoiding cumbersome processes

Active Publication Date: 2019-08-02
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, Chikungunya fever has become a recurrent infectious disease that seriously threatens human health, but there is still no effective treatment drug and preventive vaccine

Method used

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  • A kind of dna molecule comprising the whole genome cdna of chikungunya virus and its use
  • A kind of dna molecule comprising the whole genome cdna of chikungunya virus and its use
  • A kind of dna molecule comprising the whole genome cdna of chikungunya virus and its use

Examples

Experimental program
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Effect test

Embodiment 1

[0034] This example is used to illustrate the method of the present invention for constructing a full-length cDNA clone of the chikungunya virus genome.

[0035] (1) DNA fragment F1 and DNA fragment F2 are obtained by chemical synthesis, wherein the sequence of DNA fragment F1 is composed of a promoter sequence and the sequence shown in nucleotides 1-7733 of SEQ ID NO: 1, and DNA fragment F2 The sequence consists of the sequence shown in nucleotides 7310-11774 of SEQ ID NO: 1, polyadenylation sequence and specific sequence for PCR identification;

[0036] (2) Use Sbf I and Sap I to digest the DNA fragment F1, so that the digested DNA fragment F1 is inserted between the Sbf I restriction site and the Sap I restriction site of the plasmid pUC18 to obtain pUC18-5 , using Sbf I and Sap I to digest the DNA fragment F2, thereby inserting the digested DNA fragment F2 between the Sbf I restriction site and the Sap I restriction site of the plasmid pUC18 to obtain pUC18-3. pUC18-5 and...

Embodiment 2

[0039] This example is used to illustrate the method of rescuing chikungunya restorer virus by using full-length cDNA clone.

[0040] (1) In vitro transcription of the full-length cDNA clone of the chikungunya virus genome

[0041] Plasmid pCHIKV-FL was extracted with a plasmid mid-prep kit (product of Qiagen), digested with Sac I and then extracted with phenol-chloroform. The linearized plasmid was quantified and aliquoted, then frozen at -20°C until use. Using the linearized full-length cDNA cloning plasmid as a template, SP6 RiboMAX Express Large ScaleRNA Production Systems (Promega company product) was used for in vitro transcription according to the kit instructions. Further, in accordance with the operating instructions of the RNeasy Mini Kit (product of Qiagen), the in vitro transcript RNA was purified and quantified, and then frozen and stored at -80°C in separate devices until use.

[0042] (2) Rescue of chikungunya recovery virus

[0043] Use liposome Lipofectamin ...

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Abstract

The present invention discloses a DNA molecule having a nucleotide sequence represented by SEQ ID NO:1, and uses thereof, and further discloses a Chikungunya virus DRDE-06 strain labeling method, recombinant plasmid containing the DNA molecule, a construction method of the recombinant plasmid, infectious clone of the Chikungunya virus having the genomic RNA corresponding cDNA nucleotide sequence represented by SEQ ID NO:1, and a construction method of the infectious clone. According to the present invention, the DNA molecule or the recombinant plasmid can be used for constructing the Chikungunya virus infectious clone having the performance similar to the wild-type virus.

Description

technical field [0001] The present invention relates to a DNA molecule and its application, in particular to a DNA molecule comprising the whole genome cDNA of Chikungunya virus, a method for marking Chikungunya virus DRDE-06 strain, and a recombinant plasmid containing the DNA molecule and its construction method, the infectious clone of chikungunya virus and its construction method. Background technique [0002] Chikungunya virus (CHIKV) belongs to the Togaviridae genus Alphavirus, members of which also include Ross River viruses, Semliki forest viruses virus) and Sindbis virus (Sindbis virus) and so on. The main vectors of CHIKV are Aedes aegypti and Ae. albopictus, which can cause chikungunya fever, which is characterized by fever, muscle pain and arthralgia. Although the mortality rate of chikungunya is low, its spread has rapidly expanded in the past 50 years, and it has spread rapidly from East Africa, where it was initially endemic, to Europe, Asia and Oceania. Es...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/34C12N15/63C12N15/66C12N7/01C12Q1/70C12Q1/6869C12R1/93
Inventor 李晓峰秦成峰邓永强姜涛
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI