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Feline parvovirus VP2 protein and prepared virus-like particles

A feline parvovirus, virus-like technology, applied in the field of genetic engineering, to increase antibody titers and prevent infection

Active Publication Date: 2022-07-22
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no specific drug and effective treatment for FPV in the market. Clinically, immune prevention is the main method, but there is no FPV vaccine approved for marketing in China.

Method used

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  • Feline parvovirus VP2 protein and prepared virus-like particles
  • Feline parvovirus VP2 protein and prepared virus-like particles
  • Feline parvovirus VP2 protein and prepared virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Amplification and sequence analysis of VP2 gene

[0020] In 2018, the suspected feline disease materials were collected and processed, and the disease materials were routinely separated and identified. It was determined that the cat leukocytes were used for HA detection with pig red blood cells, and the hemagglutination value was 7log2. HI test with known feline parvoserum showed poor cross-reactivity; evidence of a mutation in the pathogenic feline parvovirus strain.

[0021] 1. Amplification of cat small VP2 gene

[0022] Primers were designed and synthesized according to the cat small VP2 gene sequence published in NCBI. The sequence information of the primers is as follows:

[0023] primer1: 5′-ATGAGTGATGGAGCAGTTCAACC-3′;

[0024] primer2: 5'-TTAATATAATTTTCTAGGTGCTAGTTG-3'.

[0025] The isolated cat small nucleic acid was extracted and used as a template, and primers primer1 and primer2 were used to amplify the target fragment by PCR. After sequence de...

Embodiment 2

[0029] Example 2. Construction of bacmid expressing VP2 gene

[0030] 2.1 Enzyme cleavage reaction

[0031] 2.1.1 Mark the 1.5mL EP tube to be used, add and mix the sample in the 1.5mL EP tube according to the following table: The reaction system is 50μL, and the sample is added as shown in the table below:

[0032]

[0033]

[0034] 2.1.2 Place the 1.5mL EP tube in step 2.2.1 in a constant temperature water bath at 37°C for 2-3h.

[0035] 2.1.3 Double-enzyme digestion product gel recovery

[0036] The above-mentioned double-enzyme digestion system was taken out and subjected to agarose gel electrophoresis to recover the DNA fragments therein.

[0037] (1) Label the sample collection EP tube, adsorption column and collection tube.

[0038] (2) Weigh the marked empty EP tube and record the value.

[0039] (3) Carefully cut a single target DNA band from the agarose gel with a scalpel on a gel cutter and put it into a clean 1.5 mL centrifuge tube.

[0040] (4) Add 600 ...

Embodiment 3

[0100] Example 3SF9 cell transfection

[0101] (1) Preparation: UV sterilization in a biological safety cabinet for 30 minutes; TNM-FH culture solution was placed in a 27°C water bath and preheated to 27°C.

[0102] (2) Add 2 μg of recombinant DNA to 100 μl of serum- and double-antibody-free TNM-FH medium, and mix well. Add 9 μl of Cellfectin Reagent to 100 μl of serum- and double-antibody-free TNM-FH medium, and mix well. The liposomes were mixed with recombinant DNA and left at room temperature for 40 min.

[0103] (3) Remove the cells from the 6-well plate from the 27°C incubator, discard the supernatant medium, wash the cells three times with pre-warmed TNM-FH medium, and discard the TNM-FH medium.

[0104] (4) Add 2 ml of TNM-FH medium containing 10% fetal bovine serum to each cell well.

[0105] (5) Gently add the mixture of recombinant DNA and liposome to each well of cells, mix gently, and incubate at 27°C for 5-6 hours.

[0106] (6) Discard the liquid in the well,...

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Abstract

The invention provides the feline parvovirus VP2 protein and the prepared virus-like particles, wherein the amino acid sequence of the VP2 protein is SEQ ID NO:2; the nucleotide sequence of its coding gene is SEQ ID NO:1. Another aspect of the present invention provides a recombinant baculovirus, which contains the nucleotide fragment encoding the above-mentioned VP2 protein; the recombinant baculovirus constructed in the present invention is used to prepare virus-like particles of feline parvovirus in insect cells. The invention also provides a feline parvovirus subunit vaccine, which includes an antigen and a vaccine adjuvant, and the antigen used is the virus-like particle prepared by the invention. The vaccine prepared by the invention can increase the antibody titer of the cat after immunizing the cat, and can effectively prevent the infection of the feline parvovirus.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more specifically relates to a feline parvovirus VP2 protein, and a virus-like particle (VLP) expressing feline parvovirus with an insect cell expression system; also relates to the obtained recombinant virus-like particle, and Application in the development of feline parvovirus vaccine. Background technique [0002] Feline panleukopenia is a highly contagious acute infectious disease caused by feline parvovirus (Feline panleukopenia virus, FPV), with high morbidity and mortality. Affected animals showed symptoms such as high fever, vomiting, diarrhea, severely reduced white blood cell count, and enteritis. The virus infects many species of cats and mustelids under natural conditions, such as tigers, leopards, lions and raccoons, but smaller cats, including mink, are most susceptible. The disease brings significant economic losses to the animal farming industry every year. [0...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/015C12N15/35C12N7/01A61K39/23A61P31/20
CPCC07K14/005C12N7/00A61K31/12A61P31/20C12N2750/14022C12N2750/14023C12N2750/14021C12N2750/14034A61K2039/5258A61K2039/552
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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