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DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain, as well as construction method and application thereof

An infectious cloning, encephalitis virus technology, applied in the field of reverse genetics technology and RNA virus rescue, can solve problems that have not yet been seen, and achieve good infectivity

Inactive Publication Date: 2013-05-08
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] At present, there are no domestic and foreign reports on the construction of DNA-based infectious clones of the Japanese encephalitis virus SA14-14-2 vaccine strain

Method used

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  • DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain, as well as construction method and application thereof
  • DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain, as well as construction method and application thereof
  • DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain, as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Genome sequence identification of embodiment 1 Japanese encephalitis virus vaccine strain SA14-14-2

[0049] 1. Obtaining Genome RNA of Live Attenuated Vaccine SA14-14-2 Strain Virus

[0050] (1) Take a freeze-dried preparation of Japanese encephalitis live attenuated vaccine (batch number 200710049-2, SA14-14-2 virus content: 5.4 lgPFU), add 0.5mL virus diluent to reconstitute.

[0051] (2) Extraction of viral genome RNA: Add 0.8ml Trizol reagent to 0.5ml vaccine diluent, mix well, and let stand at room temperature for 5 minutes; add 0.2ml chloroform, mix well, let stand at room temperature for 5 minutes; centrifuge at 4°C, 12000g×20min.

[0052] (3) Take the upper aqueous phase, transfer it to a new centrifuge tube free from RNase contamination, add an equal volume of isopropanol for extraction, and let stand at room temperature for 10 minutes. Centrifuge at 4°C, 12000g×20min.

[0053] (4) Discard the supernatant, add 0.5ml of 75% alcohol to rinse, mix well and let...

Embodiment 2

[0066] Example 2 Transformation of RNA-based JEV infectious clones into DNA-based JEV infectious clones

[0067] 1. Transformation of Genome 5' Half Molecule Cloning of SA14-14-2 Vaccine Strain

[0068] Plasmid pBRKpn-J1J2 (Ying, H., 2003) is the 5' semi-molecular clone of the SA14-14-2 vaccine strain, including the 1-5581 nucleotide sequence of the genome. On this basis, the CMV sequence was merged by fusion PCR technology (SEQ ID NO: 27) was fused with the 5' end sequence of the SA14-14-2 genome, cloned into the plasmid vector pBRKpn-J1J2 through the MluI and ClaI restriction sites, and the two chimeric introns were further combined by fusion PCR technology The sequence (SEQ ID NO: 28) was inserted into the JEV genome at positions 356 and 2217 to construct plasmid pCMV-J1J2. In addition, according to the sequencing results in Table 1, site-directed back-mutation was performed on individual nucleotide mutations present on the plasmid pCMV-J1J2.

[0069] 2. Transformation o...

Embodiment 3

[0083] Example 3 Evaluation of the infectivity of SA-14-14-2DNA-based full-length clone pQSINGZ at the cell level

[0084] In order to evaluate the infectivity of the SA-14-14-2DNA-based full-length clone in vitro, the purified plasmid was transfected into BHK-21 cells, and the production of recovered virus was observed.

[0085] (1) Purification and transfection of pQSINGZ-EG

[0086] The SA14-14-2 full-length clone plasmid pQSINGZ-EG containing the reporter gene EGFP was extracted and purified with the Plasmid Max Kit (Plasmid Max Kit, Qiagen Company), and the concentration of the plasmid was adjusted to 1 μG / μL. Lipofectamine TM 2000, Invitrogen Company) method to transfect BHK-21 cells, every 24 hours after transfection, observe and record under the fluorescence microscope ( Figure 6 ).

[0087] (2) Purification and transfection of pQSINGZ

[0088] The SA14-14-2 full-length cloning plasmid pQSINGZ was extracted and purified with the Plasmid Max Kit (Plasmid Max Kit, Q...

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Abstract

The invention relates to a DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain and a construction method of the infectious clone. The infectious clone is constructed by adopting a pBR322 plasmid as a framework vector and then inserting the full-length cDNA of the Japanese encephalitis virus SA14-14-2 vaccine strain; the 5' end of the full-length cDNA of the SA214-14-2 vaccine strain is connected with a CMV (Cucumber Mosaic Virus) promoter, a BGH (Bovine Growth Hormone) polyadenylation sequence is added at the 3' end, and gomphosis intron sequences for stabilizing are respectively added on the 356 locus and the 2217 locus of the genome cDNA. The invention also provides application of infectious clone serving as a novel viral vector, and thus a solid foundation is provided for developing a plurality of novel vaccines for preventing and treating tumours and viral diseases.

Description

technical field [0001] The invention relates to reverse genetics technology and RNA virus rescue technology, in particular to the DNA-based infectious clone of Japanese encephalitis virus SA14-14-2 strain, its construction method and application. Background technique [0002] Yellow fever viruses belong to positive-strand RNA viruses. This genus contains more than 80 members, and more than half of them can induce diseases in humans and animals. It poses a great threat to human health (Monath TP, 1996), such as JEV, dengue fever virus (DEN), yellow fever virus (YFV), tick-borne encephalitis virus (TBEV) and so on. For a long time, the development of related vaccines and drugs has been very slow. So far, only YFV, JEV, and TBEV have vaccines, while most of the other members have no effective prevention and treatment methods. [0003] Epidemic Japanese encephalitis (JE) is the most common type of viral encephalitis in Asia. my country is the country with the largest number of ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N15/86C12N7/00C12N7/01A61K39/12A61K39/295A61P31/14A61P35/00C12R1/93
Inventor 黄莺贾丽丽孙志伟徐宏山俞炜源俞永新
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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