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Preparation method and application of virus-like particles of dengue viruses

A dengue virus, virus-like technology, applied in the field of genetic engineering, can solve problems such as virus-like particles of type 4 dengue virus that have not yet been found

Inactive Publication Date: 2011-01-19
中国疾病预防控制中心病毒病预防控制所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, domestic research has not found any reports on the secretion and expression of dengue virus type IV virus-like particles in eukaryotic systems

Method used

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  • Preparation method and application of virus-like particles of dengue viruses
  • Preparation method and application of virus-like particles of dengue viruses
  • Preparation method and application of virus-like particles of dengue viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 Transformation of DENV1-4prME gene element

[0047] 1. DENV1-4, JEV culture and total RNA extraction

[0048] The DENV-1 GZ01 / 95 strain, DENV-2 ZS01 / 01 strain, DENV-3 H87 strain, DENV-4 H241 strain, and JEV SA14-14-2 strain (Chengdu Institute of Biological Products) were inoculated on the patch respectively. For the C6 / 36 cells with full wall, when the cell lesion reaches +++~++++, the total RNA of the cells is extracted by the Trizol reagent method. Trizol LS reagent is a product of Invitrogen, USA.

[0049] 2. Transformation of DENV1-4 prME gene elements

[0050] Using ThermoScript TM The RT-PCR System (Invitrogen, USA) reverse-transcribed the RNA of DENV 1-4 and JEV to synthesize the first strand of cDNA. The prM-E gene of DENV1-4 was modified by fusion PCR: (1) The first round of PCR amplified the signal peptide sequences JESS, DENV1-4 prME, and DENV1 from the Japanese encephalitis virus SA14-14-2 strain respectively -4prMEΔ20% and 20% of the C-...

Embodiment 2

[0111] Example 2 Construction and identification of recombinant vector capable of secreting and expressing DENV1-4 VLPs

[0112] The prME gene transformed by fusion PCR in Example 1 was subjected to double digestion with NheI and NotI to recover the target fragment, and was directionally ligated with the pcDNA5 / FRT vector (product of Invitrogen Company) through the same double digestion, and the ligation product was transformed into E.coli DH5α (product of Stratagene, USA) Competent cells were picked and inoculated into LB medium containing antibiotics for 12 hours of shaking culture at 37°C. The plasmid was digested and sequenced for identification after a small amount of preparation. The plasmids that were identified correctly were selected and named as:

[0113] pJD1prME, pJD1prMEΔ20%, pJD1prMEΔ20%JEV;

[0114] pJD2prME, pJD2prMEΔ20%, pJD2prMEΔ20%JEV;

[0115] pJD3prME, pJD3prMEΔ20%, pJD3prMEΔ20%JEV;

[0116] pJD4prME, pJD4prMEΔ20%, pJD4prMEΔ20%JEV;

[0117] The schemat...

Embodiment 3

[0124] Example 3 Preparation, Identification and Immunogenicity Experiment of DENV1-4 VLPs

[0125] 1. Preparation and identification of DENV1-4 VLPs

[0126] Harvest 2L each of pJD1prME and pJD1-4prMEΔ20% JEV transfection supernatants, high-power concentration to 3ml, and then use 15-60% sucrose density gradient centrifugation to purify DENV1-4 VLPs, extract the bands of each layer for SDS-PAGE and Western blot detection. Figure 4 It is the SDS-PAGE identification map of DENV-1 and DENV-2 VLPs, Figure 5 It is the Western blot identification diagram of DENV-1 and DENV-2 VLPs.

[0127] 2. Preliminary immunogenicity experiment of DENV-1 and DENV-2 VLPs

[0128] 1. Immunization scheme: 4-6 weeks old Balb / c mice were injected with DENV-1 VLPs or DENV-2 VLPs on days 0, 7, and 28, and 1×PBS was used as a negative control.

[0129] 2. ELISA detects serum anti-rEIII protein antibody (rEIII protein is the EIII protein of four types of dengue viruses expressed in series in a proka...

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Abstract

The invention relates to a preparation method and application of virus-like particles (VLPs) of dengue viruses. The preparation method comprises the steps of: respectively modifying four types of dengue viruses (DENV) prM-E gene elements by means of a fusing PCR (Polymerase Chain Reaction) technology, then respectively cloning the four types of modified dengue viruses prM-E gene elements into a eukaryon system expression vector, respectively transfecting a mammalian cell with the recombinant expression vectors, and secreting the mammalian cells to express the virus-like particles of the dengue viruses. In addition, the invention lays a foundation for the development of a dengue viruses VLPs polyvaccine by carrying out preliminary study on the immune effects of DENV-1 VLPs and DENV-2 VLPs.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a preparation method and application of dengue virus virus-like particles. Background technique [0002] Dengue virus (DENV) infection is the most prevalent arbovirus disease in humans today. Dengue virus is transmitted by Aedes mosquitoes and is now distributed in almost all tropical and subtropical regions. According to WHO estimates, there are 2.5 to 3 billion people in the world who are at risk of being infected with dengue virus, and about 50 to 100 million cases of dengue fever (Dengue fever DF) occur every year. Patients show high fever, headache, muscle pain, joint pain and Skin rash and other symptoms; 500,000 of them developed into more serious dengue hemorrhagic fever (Dengue hemorrhagic fever DHF) and dengue shock syndrome (Dengue shock syndrome DSS), and the number of deaths due to dengue virus infection is 2.5 per year Ten thousand. In recent years, with the gl...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N15/85C12N5/10C12N7/04A61K39/12A61P31/14
CPCY02A50/30
Inventor 李德新梁米芳张硕李川苗芳顾雯
Owner 中国疾病预防控制中心病毒病预防控制所
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