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Hybridoma cell 3A6 strain capable of secreting anti-feline parvovirus VP2 protein monoclonal antibody and application of hybridoma cell 3A6 strain

A monoclonal antibody and feline parvovirus technology, which is applied to antiviral immunoglobulins, antiviral agents, and medical preparations containing active ingredients, can solve the problems of uneven antibody neutralization potency, high preparation cost, and easy Spread other viruses and other problems, to achieve the effect of excellent biological performance and remarkable therapeutic effect

Active Publication Date: 2021-03-26
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Feline parvovirus hyperimmune serum is often used to treat feline parvovirus disease, but the preparation cost is high, it is easy to spread other viruses, and the antibody neutralization titer is uneven, so it is difficult to standardize

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Establishment of Monoclonal Antibody Hybridoma Cell 3A6 Strain

[0024] 1. Preparation of FPV antigen

[0025] Inoculate the current popular FPV virulent LYG-18 strain (stored in the Bioveterinary Medicine Laboratory of the Veterinary Research Institute of the Jiangsu Academy of Agricultural Sciences, the virus genome sequence information has been uploaded to the GenBank database, number: MW017574) into FK81 cells (purchased from the ATCC cell bank) , after the cells were completely damaged, the supernatant was collected, freeze-thawed three times, and centrifuged at 8000r / min for 3 minutes to remove cell debris; the supernatant was collected and added with PEG 6000 (product of Sigma Company) to a final concentration of 9%, then sodium chloride was added to a final concentration of 3%, stirred at room temperature until dissolved, and precipitated overnight at 4°C. The next day, centrifuge at 8000r / min for 1 hour, discard the supernatant, and resuspend with 5m...

Embodiment 2

[0042] The biological characteristic of embodiment 2 monoclonal antibody

[0043] 1. Chromosomal analysis of hybridoma cell lines

[0044] Chromosome counts were performed on hybridoma cells by Giemsa staining. Take SP2 / 0 myeloma cells and positive hybridoma cells for culture, grow to the logarithmic phase, add colchicine to the cell bottle to make the final concentration 0.1 μg / ml, and then put them in the cell culture box to continue culturing for 4 ~5 hours. Use 5 mL of 0.075mol / L KCI hypotonic solution pre-warmed at 37°C to blow up the cells and mix them evenly, place them in a 37°C incubator for 30 minutes, and add a freshly prepared fixative solution (containing methanol and glacial acetic acid, methanol: glacial acetic acid The volume ratio is 3:1) 1mL, mix well while adding dropwise, and centrifuge at 1000r / min for 10 minutes. Discard the supernatant and keep the cell pellet, blow up the cells with 5mL fixative solution, act at 37°C for 30 minutes, centrifuge at 100...

Embodiment 3

[0055] The curative effect test of embodiment 3 monoclonal antibody therapeutic agent

[0056] 1. Preparation of monoclonal antibody 3A6 therapeutic agent

[0057] Monoclonal antibody 3A6 was prepared by cell culture supernatant method. The hybridoma cell 3A6 strain secreting anti-feline parvovirus VP2 protein specific monoclonal antibody was cultured with RPMI-1640 medium containing 10% FCS (product of Thermo Fisher Scientific) in a cell culture flask, and the cells were treated Density grows to 80%-90% (cell concentration is about 2×10 6 cells / mL), replace the medium with serum-free RPMI-1640 medium, place in a 5% CO2 incubator and cultivate until all cells die, centrifuge the culture solution at 1000r / min for 10 minutes, and take the supernatant at -20°C Save for later.

[0058] 2. Clinical application of monoclonal antibody 3A6 therapeutic agent

[0059] The prepared monoclonal antibody 3A6 therapeutic agent was used for the treatment of cats with feline parvovirus dis...

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PUM

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Abstract

The invention discloses a hybridoma cell 3A6 strain capable of secreting an anti-feline parvovirus VP2 protein monoclonal antibody and an application of the hybridoma cell 3A6 strain, and belongs to the field of biology. The hybridoma cell 3A6 strain is preserved in China Center for Type Culture Collection on December 10, 2020, and the preservation number is CCTCC NO: C2020248. The hybridoma cell3A6 strain can react with eukaryotic expression VP2 protein and feline parvovirus, the biological performance is very excellent, the neutralizing titer of the prepared mouse ascites monoclonal antibody to virus is as high as 5 * 108, the reaction titer to VP2 protein is as high as 108, the neutralizing capacity to various feline parvovirus strains is equivalent, and the hybridoma cell 3A6 strain is used for clinical treatment of feline parvovirus infected cats, and the effective rate reaches 100%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically discloses a hybridoma cell 3A6 strain secreting a monoclonal antibody against feline parvovirus VP2 protein and an application thereof. Background technique [0002] Feline parvovirus disease, also known as feline distemper and feline panleukopenia syndrome, is a severe infectious disease caused by feline parvovirus (FPV), characterized by severe vomiting, diarrhea, and foul-smelling feces. The disease is characterized by high incidence, strong infectivity, rapid course of disease, and high mortality of young cats. It is one of the most important infectious viral diseases of cats. FPV is a non-enveloped single-stranded negative-sense DNA virus of the genus Parvovirus in the family Parvoviridae, with a genome length of 5.2 kb, encoding two nonstructural proteins, NS1 and NS2, and three structural proteins, VP1, VP2 and VP3. The antigenic epitope of the virus is usually located on its...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/08A61K39/42A61P31/20C12R1/91
CPCC07K16/081A61P31/20C07K2317/94C07K2317/76A61K2039/505A61K2039/552A61K2039/545
Inventor 夏兴霞毕振威钱晶王永山王晶宇诸玉梅谭业平欧阳伟王晓丽马孙婷
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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