DNA molecule, recombinant virus and preparation method and application of DNA molecule and recombinant virus

A DNA molecule and recombinant virus technology, applied in the biological field, can solve problems such as the decline of mosquito vector control, and achieve the effect of simple method, saving time and manpower, and strong proliferation ability

Active Publication Date: 2022-05-27
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(3) Mosquito vector control efforts have declined, and mosquito vectors of yellow fever virus have invaded urban environments
However, there is no report on the recombinant yellow fever virus with Nano luciferase as the reporter gene

Method used

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  • DNA molecule, recombinant virus and preparation method and application of DNA molecule and recombinant virus
  • DNA molecule, recombinant virus and preparation method and application of DNA molecule and recombinant virus
  • DNA molecule, recombinant virus and preparation method and application of DNA molecule and recombinant virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0064] The recombinant vector can be either a recombinant cloning vector or a recombinant expression vector. According to an embodiment of the present invention, the recombinant vector may be a recombinant vector in which the DNA molecule is inserted between the multiple cloning sites (such as NotI and Xho I) of the pANCR vector (the sequence is shown in SEQ ID NO: 4). .

[0065] The transgenic cell line can be a cell containing the recombinant vector of the present invention, for example, can be obtained by transferring the recombinant vector of the present invention into cells (eg, BHK-21 cells or Vero cells).

[0066] The recombinant strain can be a strain containing the recombinant vector of the present invention, for example, it can be obtained by transforming the recombinant vector of the present invention into a competent strain (eg, Escherichia coli competent strain MC1061).

[0067] The recombinant virus of the present invention can be obtained by introducing the rec...

Embodiment 1

[0073] This example is used to illustrate the construction and identification of recombinant yellow fever virus Nluc-YF-17D.

[0074] 1. Construction of recombinant plasmid pACNR-NLuc-YF-17D

[0075] (1) The full-length infectious clone pACNR / FLYF-17Dx of the yellow fever virus vaccine strain YF-17D (gifted by Prof. Charles M. Rice of The Rockefeller University, Bredenbeek PJ, Kooi EA, Lindenbach B, Huijkman N, RiceCM, Spaan WJ .2003.A stable full-length yellow fever virus cDNA clone and therole of conserved RNA elements in flavivirus replication.The Journal ofgeneral virology 84:1261-1268.) and pNL1.1 plasmid (purchased from Premega) as templates, passed Oligo7 The software designed PCR primer sequences, amplified the Nluc gene fragment with ordinary PCR, and inserted the Nluc reporter gene into the full-length infectious clone pACNR / FLYF-17Dx of the yellow fever virus vaccine strain YF-17D by multiple fusion PCR, and constructed a Yellow fever reporter virus full-length inf...

Embodiment 2

[0117] This example is used to illustrate the use of IFA to detect the viral protein expression of recombinant yellow fever virus in BHK-21 cells. The following determinations were made to the recombinant yellow fever virus and yellow fever attenuated vaccine strain YF-17D obtained in the section "Second, the rescue of recombinant yellow fever virus Nluc-YF-17D" in Example 1:

[0118] 1. Transfer BHK-21 cells to a 6-well plate pre-placed with a coverslip, at 37°C, 5% CO 2The cells were cultured in an incubator until the cell coverage area reached about 80%, and BHK-21 cells were infected with the rescued recombinant yellow fever virus and yellow fever attenuated vaccine strains respectively. It was fixed in acetone for 30 minutes, washed with PBS and air-dried to obtain an antigen sheet, which was then stored in a -20°C refrigerator.

[0119] 2. Detect the virus-specific protein in BHK-21 cells by indirect immunofluorescence, the method is as follows: take out the antigen she...

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Abstract

The invention relates to the technical field of biology, and discloses a DNA molecule and a preparation method and application thereof, and a recombinant virus and a preparation method and application thereof. The DNA molecule contains a 5'non-coding region sequence, a coding sequence of a capsid protein C into which a coding sequence of Nano luciferase is inserted, a coding sequence of a membrane and a membrane protein precursor prM, a coding sequence of an envelope protein E, a coding sequence of a non-structural protein and a 3 'non-coding region sequence which are connected in series, the 5'non-coding region sequence, the coding sequence of the capsid protein C, the coding sequence of the membrane and membrane protein precursor prM, the coding sequence of the envelope protein E, the coding sequence of the non-structural protein and the 3 'non-coding region sequence are derived from the yellow fever virus. The cDNA sequence corresponding to the genome RNA of the recombinant virus is the same as the sequence of the DNA molecule. The DNA molecule and the recombinant virus can be used for screening yellow fever virus treatment drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a DNA molecule, a recombinant virus, a preparation method and application thereof, and in particular, to a DNA molecule, a preparation method and application thereof, a recombinant virus, and a preparation method and application thereof. Background technique [0002] Yellow fever virus (YFV) belongs to the genus Flaviviridae of the family Flaviviridae and is a single-stranded positive-stranded RNA virus. Yellow fever virus is mainly prevalent in Africa and the Americas, and my country has imported cases from time to time. Humans are mainly infected with yellow fever virus through mosquito bites. Infection with yellow fever virus can lead to fatal hemorrhagic fever. The current means of preventing and controlling the epidemic of yellow fever are mainly vaccination and mosquito vector control, and there are no antiviral drugs that can be used to treat yellow fever. [0003] In recent ye...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/53C12N7/01C12Q1/02C12R1/93
CPCC12N15/85C12N9/0069C12N7/00C12Q1/025C12Y113/12013C12N2800/107C12N2770/24152C12N2770/24121C12N2770/24171G01N2333/185Y02A50/30
Inventor 秦成峰刘忠钰董好龙覃晓玲李晓峰
Owner ACADEMY OF MILITARY MEDICAL SCI
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