A kind of dengue virus degenerate vaccine and its application

A dengue virus and vaccine technology, applied in the direction of antiviral immunoglobulin, application, viral peptides, etc., can solve the problem that type 2 dengue fever virus strains cannot protect, the protection effect of specific epidemic strains is not good, and the overall effect is not ideal and other problems, to achieve the effects of low mammalian cell expression system, cost control, and antibody-dependent enhancement

Inactive Publication Date: 2014-10-22
ARMY MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Indian Etemad B et al. (2008) and Chinese scholar Chen S et al. (2007) fused the specific regions of the E protein of 4 serotypes of dengue virus together to construct a 4-valent recombinant protein vaccine, which has a certain protective effect at the animal level. And the titer is 1:47-1:588, the protective power of various types of DV is quite different, which is not conducive to popularization
[0007] Bowen et al. (2012) in the United States disclosed a method of using a computer to design a physically and chemically consistent EDIII protein-binding antibody based on the EDIII protein sequence of 671 strains of DV. Although the antibody produced has a wide range of protection, it has poor specificity and may be specific to The protection effect of epidemic strain infection is not good, which is not conducive to the promotion
[0008] The first dengue vaccine reported by French Sabchareon et al. (2012)—CYD-TDV recombinant attenuated tetravalent dengue virus vaccine can be immune to dengue virus strains 1, 3 and 4, but its overall effect is not ideal. Type 2 strain of dengue virus most common in Thailand offers no protection

Method used

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  • A kind of dengue virus degenerate vaccine and its application
  • A kind of dengue virus degenerate vaccine and its application
  • A kind of dengue virus degenerate vaccine and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Design of Dengue Virus Degeneracy Sequence Vaccine Molecules

[0045] 1. Sequence Alignment

[0046] DV is widely popular in tropical and subtropical regions, especially in Southeast Asia, Africa, Central and South America and the Western Pacific. The present invention selects DV strains that have been popular in the world, and the DV strains include representative strains of four serotypes of DV-1, DV-2, DV-3 and DV-4. DV-1 selects the epidemic strains in the Southwest Indian Ocean and Africa (GenBank accession number DQ285558), the Indonesian epidemic strains (GenBank accession number AB189121), the American strains (GenBank accession number GQ868530) and the Chinese epidemic strains (GenBank accession number AY376738); DV-1 2 Select the TR1751 strain popular in the United States and Japan (Trent, et al.1983), the popular strain in West Africa (GenBank accession No. EF105386 and EF105378), the American strain (GenBank accession No. HM582117); DV-3 selec...

Embodiment 2

[0063] Example 2 Construction of Dengue Virus Degenerate Vaccine Recombinant Expression Plasmid

[0064] 1. BamHI (TaKaRa Company) and XhoI (TaKaRa Company) double enzyme digestion of chemically synthesized degenerate sequence DVIII

[0065]

[0066] After mixing, put it in a water bath at 37°C for 4 hours, run 1.2% agarose (Shanghai Sangong) gel electrophoresis, and recover the double-digested DVIII fragment according to the instructions of the Promega gel recovery kit;

[0067] 2. The prokaryotic expression vector pET22b was double-digested by BamHI (TaKaRa Company) and XhoI (TaKaRa Company), and the double-enzyme digestion reaction system was the same as above, and the plasmid was recovered;

[0068]

[0069] After mixing, put it in a water bath at 16°C for 16 hours, and inactivate the ligase at 65°C for 10 minutes;

[0070] 4. Transformation experiments

[0071] For the preparation of Escherichia coli DH5α competent cells, refer to the Molecular Cloning Experime...

Embodiment 3

[0072] Example 3 Screening and Characterization of Recombinant Bacterial Clones

[0073] 1. Transform the recombinant expression plasmid pET22b-DVIII described in Example 2 into a competent BL21 Escherichia coli according to the Molecular Cloning Experiment Guide (Third Edition, Science Press, 2002). For details, refer to the description in step 4 in Example 2 method is carried out.

[0074] 2. Pick 10 AMP-resistant clones and culture them with 1ml LB medium. When the OD600 value is about 0.3-0.5, add 0.5mM IPTG (Beijing Saibaisheng) inducer to induce for 1 hour, and centrifuge at 14000 rpm for 5 Minutes to collect the cells.

[0075] 3. Carry out SDS-PAGE electrophoresis (Bio-Rad, USA) according to the method of the molecular cloning experiment guide, and the gel after electrophoresis is stained with Coomassie Blue (Fluka, Switzerland). The clones with clear expression bands were expression-positive clones, named as pET22b-DVIII / BL21 engineering bacteria.

[0076] 4. Pu...

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Abstract

The invention relates to a dengue virus degeneration vaccine and application thereof. The amino acid sequence of the dengue virus degeneration vaccine is shown as SEQ ID NO.2; the amino acid sequence of the dengue virus degeneration vaccine contains an antigen peptide fragment, a dengue virus envelope glycoprotein EDIII region, a virus virulence related gene neutralizing epitope, a beta lamella and Loop amino acid, wherein the antigen peptide fragment is determined after sequence comparison analysis is carried out on a dengue virus representative strain which respectively contains serotypes 1,2,3,4; and the beta lamella retains a basic structure which forms the dengue virus envelope glycoprotein EDIII region. The dengue virus degeneration vaccine disclosed by the invention can be used for immunizing an organism, stimulating the organism to generate humoral immunity response and preventing the dengue virus infectious diseases.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a dengue virus degenerate vaccine and its preparation method and application. Background technique [0002] Dengue virus (DV) is an enveloped single-stranded positive-sense RNA virus belonging to the genus Flaviviridae. It uses mosquitoes as a vector and is widely prevalent in tropical and subtropical regions. Human dengue fever (classical dengue fever, DF) and dengue hemorrhagic fever / dengue shock syndrome (dengue hemorrhagic fever / dengue shock syndrome, DHF / DSS) caused by DV infection seriously endanger human health. There are 2.5 to 3 billion people living in endemic areas in the world, and more than 100 million people are infected every year. DHF cases are about 500,000, and the mortality rate is 5 to 20%. If the treatment is not timely, it can reach 50%. In recent years, with many reasons such as global warming, population flow, ecological environment deteri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/18C07K16/10C12N15/40C12N15/70C12N1/21A61K39/12A61P31/14
CPCY02A50/30
Inventor 饶贤才杨杰胡珍胡晓梅陈炜
Owner ARMY MEDICAL UNIV
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