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Preparation method and application of double-effect vaccine for dengue fever

A technology of dengue virus and protein is applied in the field of preparation of dengue virus double-effect vaccine, which can solve the problems of ineffective treatment and safe and effective dengue vaccine, and achieve the effects of reducing bleeding and death, and reducing the rate of virulence.

Active Publication Date: 2016-05-25
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Dengue fever has been discovered for more than 200 years, but there is still no effective treatment and safe and effective dengue vaccine

Method used

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  • Preparation method and application of double-effect vaccine for dengue fever
  • Preparation method and application of double-effect vaccine for dengue fever
  • Preparation method and application of double-effect vaccine for dengue fever

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1, the acquisition of DENV2△NS1 protein

[0053] Express DENV2NS1 and DENV2△NS1 proteins through prokaryotic, as follows:

[0054] 1. Acquisition of DENV2NS1 gene and DENV2△NS1 gene

[0055] 1) DENV2NS1 gene

[0056] Extract the total RNA of DENV2 virus, reverse transcribe to obtain cDNA as a template, and use F1 and R1 primers to PCR amplify to obtain a 1056bp PCR product, which is the DENV2NS1 gene. After sequencing, its nucleotide sequence is sequence 1, which encodes The protein is DENV2NS1, the amino acid length of the protein is 352AA, and the amino acid sequence is sequence 2.

[0057] 2) DENV2△NS1 gene

[0058] The total RNA of DENV2 virus was extracted, and the cDNA obtained by reverse transcription was used as a template for amplification.

[0059] The first step: using the cDNA of the DENV2 virus as a template, carry out PCR amplification with the primer pair of F2 and R2, and obtain the PCR product of 1-115AA; Amplify to obtain a PCR product of ...

Embodiment 2

[0090] Example 2, the application of DENV2NS1 and DENV2△NS1 in passive immunization to hinder the acquisition of dengue virus

[0091] 1. Preparation of DENV2NS1 mouse-derived antiserum and DENV2△NS1 mouse-derived antiserum

[0092] 1. Preparation of mouse antiserum.

[0093] The experimental animals were 8-week-old Balb / c female mice, and the experimental procedure was as follows:

[0094] (1). On the first day (initial immunization), each mouse was injected intraperitoneally with an equal-volume mixture of DENV2NS1 and DENV2△NS1 proteins prepared in the above-mentioned one and Freund's complete adjuvant (containing 40ug of DENV2NS1 or DENV2△NS1 proteins).

[0095] (2). On the 14th day (the first booster immunization), each mouse was subcutaneously injected with an equal-volume mixture of the DENV2NS1 and DENV2△NS1 proteins prepared above and Freund's incomplete adjuvant (containing 40ug of DENV2NS1 or DENV2△NS1 proteins). ).

[0096] (3). On the 28th day (the second boost...

Embodiment 3

[0123] Embodiment 3, the application of DENV2NS1 and DENV2△NS1 as dengue fever vaccine

[0124] 1. Active immunization of AG6 mice with DENV2NS1 protein and DENV2△NS1 protein

[0125] The DENV2NS1 protein and DENV2△NS1 protein prepared in Example 1 were actively immunized in AG6 mice. The immunized mice were 6-week-old AG6 mice. The experiment was divided into three groups, PBS control group, DENV2NS1 and DENV2△NS1 experimental groups , 12 AG6 mice in each group, the immunization method is as follows:

[0126] 1. Day 1 (primary immunization)

[0127] DENV2NS1 experimental group: each AG6 mouse was injected intraperitoneally with an equal-volume mixture of DENV2NS1 prepared in Example 1 and Freund's complete adjuvant (40ugDENV2NS1 per mouse);

[0128] DENV2△NS1 experimental group: each AG6 mouse was intraperitoneally injected with an equal volume mixture of DENV2△NS1 prepared in Example 1 and Freund's complete adjuvant (40ug DENV2△NS1 per mouse);

[0129] Control group: each...

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Abstract

The invention discloses a preparation method and application of a double-effect vaccine for the dengue fever. The invention provides a protein which is protein a) or protein b, wherein protein a) is a protein composed of an amino acid sequence as shown in a sequence 4 in a sequence table, and protein b) is a protein which has same functions as protein a) and is derived from the protein a) by subjecting the sequence 4 in the sequence table to substitution and / or deletion and / or addition of one or more amino acid residues. Experiments in the invention prove that reconstructed dengue virus non-structural protein 1 (DENV delta NS1) can be used as the vaccine; and the double-effect vaccine can protect mankind or mammals from dengue haemorrhagic fever and block propagation of the dengue virus in nature via mosquitoes.

Description

technical field [0001] The invention belongs to the field of prevention and treatment of dengue virus, in particular to the preparation and application of a dengue virus double-effect vaccine. Background technique [0002] Dengue fever (Dengue Fever, DF) is an acute viral infectious disease transmitted by Aedes mosquitoes. Dengue virus (DENV) is spherical and consists of cell membrane, capsid protein, nonstructural protein and single-stranded positive-strand RNA. In nature, there are four serotypes (DENV-1-DENV-4) of dengue virus, and the nucleotide sequences of each serotype differ greatly, up to more than 35%. Within each serotype, it can be divided into multiple genotypes according to different endemic areas. After the dengue virus infects the human body through the bite of an Aedes mosquito, it can show clinical symptoms after 2-3 days of incubation. The clinical features of dengue are high fever, bone and muscle pain, bleeding tendency and significantly decreased whi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C12N15/40A61K39/12A61P31/14
CPCA61K39/12A61K2039/55566C07K14/005C12N2770/24122C12N2770/24134C07K14/18Y02A50/30
Inventor 程功刘建英刘洋
Owner TSINGHUA UNIV
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