Dengue virus degeneration vaccine and application thereof

A dengue virus and vaccine technology, applied in antiviral immunoglobulins, applications, viral peptides, etc., can solve the problem that the type 2 dengue virus strain cannot play a protective role, the protection effect of specific epidemic strains is poor, and the protective power is relatively different. Large and other problems, to achieve the effects of low mammalian cell expression system, cost control, and improved safety of use

Active Publication Date: 2013-07-10
ARMY MEDICAL UNIV
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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

Method used

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  • Dengue virus degeneration vaccine and application thereof
  • Dengue virus degeneration vaccine and application thereof
  • Dengue virus degeneration vaccine and application thereof

Examples

Experimental program
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Example Embodiment

[0044] Example 1 Design of Dengue Virus Degenerate Sequence Vaccine Molecule

[0045] 1. Sequence alignment

[0046] DV is widespread 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: DV-1, DV-2, DV-3 and DV-4. DV-1 selected epidemic strains in Southwestern Indian Ocean and Africa (GenBank accession number DQ285558), Indonesia epidemic strain (GenBank accession number AB189121), American epidemic strain (GenBank accession number GQ868530) and China epidemic strain (GenBank accession number AY376738); DV- 2 Select the TR1751 strain popular in the United States and Japan (Trent, et al.1983), the western African strain (GenBank accession numbers EF105386 and EF105378), the American epidemic strain (GenBank accession number GQ199892) and the Pacific region epidemi...

Example Embodiment

[0063] Example 2 Construction of recombinant expression plasmid of dengue virus degenerate vaccine

[0064] 1. The chemically synthesized degenerate sequence DVIII is double digested with BamHI (TaKaRa company) and XhoI (TaKaRa company)

[0065]

[0066] After mixing, place it in a water bath at 37°C for 4 hours, perform electrophoresis on 1.2% agarose (Shanghai Shenggong) gel, 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 with BamHI (TaKaRa company) and XhoI (TaKaRa company). The double digestion reaction system was the same as above, and the plasmid was recovered;

[0068]

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

[0070] 4. Conversion experiment

[0071] For the preparation of Escherichia coli DH5α competence, please refer to the Molecular Cloning Experiment Guide (third e...

Example Embodiment

[0072] Example 3 Screen and identify recombinant bacterial clones

[0073] 1. The recombinant expression plasmid pET22b-DVIII described in Example 2 was transformed into BL21 Escherichia coli competent according to the method of Molecular Cloning Experiment Guide (third edition, Science Press, 2002), please refer to the description in step 4 in Example 2 for details Method to proceed.

[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 for 1 hour and centrifuge at 14,000 rpm for 5 Minutes, collect the bacteria.

[0075] 3. Carry out SDS-PAGE electrophoresis (Bio-Rad, USA) according to the method of molecular cloning experiment guide. After electrophoresis, the gel is stained with Coomassie blue (Fluka, Switzerland). Observe that compared with the uninduced strain, there is The clone that clearly expressed the band was a positive expression clone, named pET22b-DVIII / BL21 e...

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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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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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