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A kind of dna molecule and recombinant virus and their preparation method and application

A DNA molecule and recombinant virus technology, applied in the biological field, can solve problems such as tropism diseases and nervous system side effects diseases, threats to the life safety of vaccinators, rashes, etc., and achieve stable attenuation characteristics, good application prospects, and high safety Effect

Active Publication Date: 2020-04-24
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

[0003] Although the live attenuated yellow fever vaccine YF17D is safe and effective, more and more reports point out that after vaccination, the vaccine will cause a series of side effects, including fever, headache, rash in the vaccination area, etc.
More serious ones may also cause visceral tropism and nervous system side effects with a low probability, posing a threat to the life safety of the vaccinators

Method used

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  • A kind of dna molecule and recombinant virus and their preparation method and application
  • A kind of dna molecule and recombinant virus and their preparation method and application
  • A kind of dna molecule and recombinant virus and their preparation method and application

Examples

Experimental program
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Effect test

Embodiment approach

[0041] The recombinant vector can be either a recombinant cloning vector or a recombinant expression vector. According to one embodiment of the present invention, the recombinant vector can be a recombination of the DNA molecule inserted between the multiple cloning sites (such as AscI and XhoI) of the pANCR-L1 vector (sequence shown in SEQ ID NO: 3). carrier.

[0042] 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 (such as BHK-21 cells or Vero cells).

[0043] The recombinant bacterium can be a strain containing the recombinant vector of the present invention, for example, can be obtained by transferring the recombinant vector of the present invention into a competent strain (such as Escherichia coli competent strain Top10).

[0044] The recombinant virus of the present invention can be obtained by introducing the recombi...

Embodiment 1

[0052] This example is used to illustrate the construction and identification of recombinant yellow fever virus YF-IRES.

[0053] 1. Construction of recombinant plasmid pYF-IRES

[0054] The DNA molecule shown in SEQ ID NO: 1 is inserted into the pANCR-L1 carrier (the pANCR-L1 carrier is the plasmid shown in SEQ ID NO: 3, and the 2350-2575 nucleotides from the 5' end of SEQ ID NO: 3 are sp6 promoter) between the Asc I and XhoI restriction sites to obtain the vector pANCR-YFV.

[0055] 1. Using the vector pANCR-YFV as a template, PCR amplification was performed using primers composed of NOT1 and YF17D-IRES-R1 to obtain a PCR amplification product (689bp).

[0056] NOT1: 5'-CGACGCGGCCGCGCTAGCGATGAC-3' (SEQ ID NO: 6);

[0057] YF17D-IRES-R1: 5'-GGGAGAGGGGTTAACGGCGTTTCCTTGAGGAC AATC-3' (SEQ ID NO: 7).

[0058] 2. Use the plasmid pIRES-neo (purchased from TAKARA company) as a template, and use primers composed of IRES-F and IRES-R to perform PCR amplification. The stop codon is ...

Embodiment 2

[0078] 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.

[0079] The recombinant yellow fever virus and the yellow fever attenuated vaccine strain YF17D obtained in the "two, the rescue of the recombinant yellow fever virus YF-IRES" part of Example 1 are determined as follows:

[0080] 1. Infect monolayer BHK-21 cells with the rescued recombinant yellow fever virus and yellow fever attenuated vaccine strain respectively, collect the cells 48 hours after infection, resuspend in DMEM medium containing 10% FBS, and spread on glass slides , 37°C, 5% CO 2 After culturing under the same conditions for 10 hours, the antigen sheet was obtained. The antigen sheet was fixed in acetone at -20°C for 60 minutes, dried and then sealed in a refrigerator at -20°C for use.

[0081] 2. Using the mouse immune serum of the attenuated yellow fever vaccine strain (see below for the preparation method), the vir...

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Abstract

The invention relates to the technical field of biology and discloses a DNA molecule, a recombinant virus and a preparation method and application of the DNA molecule and the recombinant virus. The DNA molecule contains a 5' noncoding region sequence, a capsid protein coding sequence, a DNA sequence, a membrane protein precursor coding sequence, an envelope protein coding sequence, a non-structural protein coding sequence and a 3' noncoding region sequence, wherein the DNA sequence corresponds to an internal ribosome entry site sequence with the 5' end connected with a termination codon sequence, and all the sequences except the DNA sequence are derived from yellow fever virus attenuated strains. The cDNA sequence corresponding to the genome RNA of the recombinant virus is identical with the sequence of the DNA molecule. The DNA molecule can be used for building the recombinant virus so as to obtain a vaccine capable of effectively preventing yellow fever virus infection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a DNA molecule, a recombinant virus, their preparation method and application, specifically, a DNA molecule, its preparation method and application, a recombinant virus, its preparation method and application, and the Recombinant virus vaccines. Background technique [0002] Yellow fever (Yellow Fever, YF) is a highly fatal hemorrhagic disease in humans and non-human primates caused by yellow fever virus (YFV). Yellow fever virus belongs to Flaviviridae family, Flavivirus genus. Its genome is a single-stranded positive-strand RNA with a length of about 11kb. The 5' end contains a type 1 cap structure, the middle is a single open reading frame, and the 3' end is a UTR region without PolyA. Through the translation pathway initiated by the classic cap structure, YF17D single ORF encodes a polyprotein, which contains three structural proteins (capsid protein C, membrane and membrane prot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/40C12N7/01A61K39/12A61P31/14
CPCA61K39/12A61K2039/5254A61K2039/541A61K2039/545C07K14/005C12N2770/24121C12N2770/24134Y02A50/30
Inventor 秦成峰李晓峰王洪江
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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