Canine parvovirus trivalence subunit vaccine

A technology of canine parvovirus and subunit vaccines, which is applied in the direction of vaccines, viruses, antiviral agents, etc., can solve the problems of poor cross-protection, strong virulence, and inability to completely prevent canine parvovirus, so as to solve simultaneous infection and improve Effect of Antibody Titer

Pending Publication Date: 2019-08-23
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since its discovery, canine parvovirus has undergone decades of evolution, and a variety of genetic variants have emerged, such as CPV-2a, CPV-2b, and CPV-2c; but CPV-2a, CPV-2b, and CPV-2c Poor cross protection, common single vaccine can not completely preve...

Method used

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  • Canine parvovirus trivalence subunit vaccine
  • Canine parvovirus trivalence subunit vaccine
  • Canine parvovirus trivalence subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Amplification and sequence analysis of VP2 gene

[0017] The suspected canine parvovirus materials collected in 2018 from 3 different regions were processed. In order to isolate the pathogen, the intestinal contents of the affected dogs were aseptically collected, centrifuged to collect the cleared bacteria, and then inoculated with cat kidney cells F81. After culturing for 5 days, the virus liquid was harvested. After the harvested virus liquid was purified, the analysis and detection of virus characteristics in terms of virus content, immunogenicity, specificity and purity were carried out. The results showed that the isolated virus strain had a specific reaction with canine parvovirus, and there was no bacteria, mycoplasma and Foreign virus contamination. The isolated 3 strains of virus were tested for HA with porcine erythrocytes, and the results were 9log2, 8log2, and 9log2 respectively. The HI test was carried out with the existing serum, and the cro...

Embodiment 2

[0026] Embodiment 2, the construction of the bacmid expressing VP2 gene

[0027] The synthesized genes of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 were digested and connected into expression vectors. And the identified positive plasmids were transferred into DH10bac to construct the recombinant bacmids of CPV-2a, CPV-2b and CPV-2c.

[0028] 2.1 Cutting and optimization of VP2 gene

[0029] 2.2 Enzyme digestion reaction

[0030] 2.2.1 Mark the 1.5mL EP tube to be used, and add and mix the sample in the 1.5mL EP tube according to the following table: the reaction system is 50 μL, and the sample addition is shown in the table below:

[0031]

[0032] 2.2.2 Place the 1.5mL EP tube in step 2.2.1 in a constant temperature water bath at 37°C for 2-3 hours.

[0033] 2.2.3 Gel recovery of double enzyme digestion products

[0034] Take out the above-mentioned double digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein.

[0035] (1)...

Embodiment 3

[0097] Embodiment 3: SF9 cell transfection

[0098] (1) Preparation: UV sterilization in a biosafety cabinet for 30 minutes; TNM-FH culture solution was placed in a 27°C water bath and preheated to 27°C.

[0099] (2) Add 2 μg of the recombinant baculovirus prepared in Example 2 to 100 μl of TNM-FH culture medium without serum and double antibody, and mix well. Add 9 μl Cellfectin Reagent to 100 μl TNM-FH medium without serum and double antibody, and mix well. The liposomes were mixed with the recombinant DNA and allowed to stand at room temperature for 40 min.

[0100] (3) Take out the 6-well plate cells from the incubator at 27°C, discard the supernatant medium, wash the cells three times with pre-warmed TNM-FH culture medium, and discard the TNM-FH culture medium.

[0101] (4) Add 2 ml of 10% fetal bovine serum TNM-FH culture solution to each cell well.

[0102] (5) Gently add the mixture of recombinant DNA and liposomes into each well of cells, mix gently, and culture st...

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Abstract

The invention provides a canine parvovirus trivalence subunit vaccine. Antigens are VP2 protein virus-like particles of a canine parvovirus and are obtained by conducting cultivation and collection after a recombinant rhabdovirus is utilized for infecting insect cells; the recombinant rhabdovirus carries nucleotide fragments for coding VP2 proteins; the amino acid sequences of the VP2 proteins areSEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively. After the prepared vaccine is utilized for immunizing a canine, the antibody titer of the canine can be increased, and infection of the canine parvovirus is prevented.

Description

technical field [0001] The invention belongs to the technical field of vaccine preparation, in particular to a trivalent subunit vaccine of canine parvovirus. Background technique [0002] Canine parvovirus mainly infects animals of the family Canidae and Felidae, especially for puppies. It is an acute and severe infectious disease, and the infection mortality rate can reach 91%. After healthy dogs are infected with the virus through the digestive tract, Symptoms of enteritis and myocarditis (A fshar A. Canine Parvovirus infection-review. Vet Bull, 1981, 51:605-612.). Enteritis type symptoms are severe vomiting and severe bloody diarrhea. Myocarditis is more common in puppies, which can lead to respiratory and cardiovascular failure in puppies. Since 1980, there have been reports of the occurrence of this disease in my country. In recent years, the problem of canine parvovirus infection has become prominent. The morbidity and fatality rate of this disease in our country a...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/866A61K39/23A61P31/20
CPCC12N7/00C12N15/86A61K39/12A61P31/20C12N2710/14043C12N2710/14021A61K2039/5258A61K2039/552C12N2750/14334C12N2750/14323
Inventor 郭伟伟刘大卫向银辉陈俭梅范根成杜元钊
Owner YEBIO BIOENG OF QINGDAO
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