Feline panleukopenia virus (FPV) VP2 protein and prepared virus-like particles

A feline parvovirus, virus-like technology, applied in the field of genetic engineering, to achieve the effect of improving antibody titer and preventing infection

Active Publication Date: 2019-08-02
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 panleukopenia virus (FPV) VP2 protein and prepared virus-like particles
  • Feline panleukopenia virus (FPV) VP2 protein and prepared virus-like particles
  • Feline panleukopenia virus (FPV) 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 parvovirus materials were collected for processing, and the disease materials were routinely separated and identified. It was determined that the feline parvovirus was tested for HA with porcine red blood cells, and the hemagglutination value was 7log2. The HI test with known feline parvovirus sera showed poor cross-reactivity; it proved that the pathogenic feline parvovirus strain had mutated.

[0021] 1. Amplification of feline parvo VP2 gene

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

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

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

[0025] Extract the isolated feline small nucleic acid as a template, and use primers primer1 and primer2 to amplify the target fragment by PCR. After sequence d...

Embodiment 2

[0029] Embodiment 2, the construction of the bacmid expressing VP2 gene

[0030] 2.1 Enzyme digestion reaction

[0031] 2.1.1 Mark the 1.5mL EP tube to be used, and add and mix the sample according to the following table in the 1.5mL EP tube: the reaction system is 50 μL, and the sample addition is 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 2-3 hours.

[0035] 2.1.3 Gel recovery of double enzyme digestion products

[0036] Take out the above-mentioned double digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein.

[0037] (1) Mark 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 out a single target DNA band from the agarose gel on a gel cutter with a scalpel and put it into a clean 1.5mL centrifuge tube.

[0040] (...

Embodiment 3

[0100] Embodiment 3 SF9 cell transfection

[0101] (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.

[0102] (2) Add 2 μg of recombinant DNA to 100 μl of TNM-FH 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.

[0103] (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.

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

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

[0106] (...

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Abstract

The invention provides feline panleukopenia virus (FPV) VP2 protein and prepared virus-like particles. The amino acid sequence of the VP2 protein is shown in SEQ ID NO: 2; the nucleotide sequence of an encoding gene of the VP2 protein is shown in SEQ ID NO: 1. In another aspect, the invention provides a recombinant baculovirus, and the recombinant baculovirus comprises nucleotide fragments encoding the VP2 protein; the constructed recombinant baculovirus is applied to preparation of the virus-like particles of the FPV in insect cells. The invention further provides an FPV subunit vaccine comprising antigens and vaccine adjuvants; the used antigens are the prepared virus-like particles provided by the invention. After being immune to a cat, the prepared vaccine provided by the invention canimprove the antibody titer of the cat, and can effectively prevent the infection of the FPV.

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 Applications(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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