Influenza compound multi-epitope DNA vaccine and application thereof

A DNA vaccine and multi-epitope technology, applied in the field of DNA vaccines, can solve the problems of unsatisfactory immune protection test results and large impact

Inactive Publication Date: 2010-01-20
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the vaccine is greatly affected by maternal antibodies to Newcastle disease, and the timing of immunization should be gras

Method used

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  • Influenza compound multi-epitope DNA vaccine and application thereof
  • Influenza compound multi-epitope DNA vaccine and application thereof
  • Influenza compound multi-epitope DNA vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The establishment of embodiment 1 influenza multi-epitope DNA vaccine

[0053] Collect the published influenza epitopes, refer to the existing research before the present invention, and download the H1, H3, H5, H7, H9 subgroups respectively from NCBI (http: / / www.ncbi.nlm.nih.gov) Amino acid sequences of HA and NA genes of influenza type 1.

[0054]Bioinformatics MHCI molecular prediction uses: web server SYPEITHI (http: / / syfpeithi.bmi-heidelberg.com), Bimas (http: / / www-bimas.cit.nih.gov / molbio / hla_bind), Multipre (http : / / antigen.i2r.a-star.Edu.sg); bioinformatics MHC class II molecule prediction uses: web server SYFPEITHI and Multipre; bioinformatics B cell epitope prediction uses: web server Bcepred (http: / / www .intech.res.in / raghava / bcepred) and biomolecular simulation software Insight II (Accelrys, 2005); proteasome cleavage prediction using: web server PAProC (http: / / www.paproc.de).

[0055] Using bioinformatics methods, the multi-subtype (H1, H3, H5, H7, H9) inf...

Embodiment 2

[0135] Example 2 Experimental Immunization Study of Mice with H3 / H1 Dominant Composite Multi-epitope Nucleic Acid Vaccine

[0136] 1. Experimental grouping, mouse immunization and sampling

[0137] Twenty BALB / c female mice were randomly divided into 2 groups, 10 mice in each group, respectively pVAX1 empty plasmid control group and pVAX-MEGNp24 plasmid immunization group. Immunization was carried out three times with an interval of 14 days. Each time, 100 μg rDNA (dissolved in 100 μL sterile saline) was injected into the bilateral tibialis anterior muscle of the mice. Blood was collected 2 weeks after the second immunization, placed overnight at 4°C, centrifuged at 5000rpm for 10min, serum was collected, and stored at -20°C for testing. On the 10th day after the third immunization, the eyeballs were picked to take blood, and the cervical spine was dislocated to kill. The serum was coagulated and separated for ELISA to detect cytokines; at the same time, the spleen was asept...

Embodiment 3

[0154] Example 3 H5 / H3 dominant compound multi-epitope nucleic acid vaccine pig experimental immunization research

[0155] 1. Experimental grouping, pig immunization and sampling

[0156] Thirty 50-day-old Landrace pigs were randomly divided into 6 groups with 5 pigs in each group. They were divided into 5 immunization groups and 1 empty plasmid control group, and the groups were marked with different ear numbers. They were immunized three times on 0d, 21d and 35d respectively. For the first immunization, 2 mg rDNA-chitosan nanoparticle suspension was intramuscularly injected into the neck of each pig. After each immunization, 1 mg rDNA was administered. Blood was collected from the hypogastric vein on 0d, 14d, 21d, 35d, and 45d, and centrifuged at 5000r / min for 10min to collect serum for ELISA antibody determination. Ten days after the third immunization, the anticoagulated blood was aseptically collected from the hypogastric vein for the separation of peripheral blood l...

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Abstract

The invention relates to influenza compound multi-epitope genes and a DNA vaccine constructed by utilizing the influenza compound multi-epitope genes. The invention adopts the method of bioinformatics, combines a network server and relevant software, forecasts HA and NA functional epitopes which are related to major protective antigens of influenza and totally obtains 17 influenza CTL epitopes from a variety of subtypes and 12 influenza Th and B cell epitopes from a plurality of subtypes. Endoplasmic reticulum (ER) guiding signals are both added in the design of two epitope boxes, thereby promoting more efficient synthesis and folding of epitope peptide. The invention further dopes a plurality of general helper T cell epitopes (HTL) in the epitope boxes in the design. The DNA vaccine constructed by the influenza multi-epitope genes can simultaneously prevent a plurality of subtype influenza virus and carry out immune protection on a variety of types of animals.

Description

technical field [0001] The invention relates to influenza vaccines, in particular to DNA vaccines based on influenza virus gene epitopes. Background technique [0002] Influenza (Influenza) is called flu for short, is an acute respiratory infectious disease caused by influenza virus (Influenza virus, IV). Influenza virus replicates in respiratory epithelial cells and causes superficial inflammation, manifesting as acute upper respiratory inflammation and symptoms of systemic intoxication. In the 1930s, humans began to recognize influenza viruses and develop vaccines. However, influenza viruses are still important pathogens that threaten human and animal health today. Over the past few years, the prevalence of influenza virus has caused huge loss of human resources and material resources. According to the different antigenicity of their internal proteins, influenza viruses can be divided into three types: A, B, and C. Type A occurs in a broad spectrum of warm-blooded anima...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/145A61P31/16
Inventor 金宁一鲁会军田明尧李昌李霄金扩世南文龙谭磊张金双赵翠青
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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