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Seneca Valley virus VP1 protein, coding gene, hybridoma cell line and monoclonal antibody and application thereof

A hybridoma cell line, monoclonal antibody technology, applied in the field of viruses, can solve the problem of ignoring foot-and-mouth disease and other issues

Active Publication Date: 2018-07-27
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as a member of the same virus family as FMDV, the prevalence of SVV may cause pig farmers to get used to pigs with symptoms similar to FMD in the future, and then ignore the possibility of FMD

Method used

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  • Seneca Valley virus VP1 protein, coding gene, hybridoma cell line and monoclonal antibody and application thereof
  • Seneca Valley virus VP1 protein, coding gene, hybridoma cell line and monoclonal antibody and application thereof
  • Seneca Valley virus VP1 protein, coding gene, hybridoma cell line and monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Antigen preparation

[0042] 1. Massive amplification of Hubei strain of Seneca Valley virus

[0043] (1) BHK-21 cells were subcultured to 175cm 2 In the culture flask, in the culture medium of DMEM+10%FBS+1%PS at 37℃, 5%CO 2 cultured to 80% fullness.

[0044] (2) Replace the medium with serum-free DMEM, add the amount of SVV-CH-HB2016 virus with an MOI of 0.1, 37°C, 5% CO 2 Under the condition of infection for 1h.

[0045] (3) Remove the supernatant and replace it with DMEM+2%FBS+1%PS medium, 37°C, 5%CO 2 Cultivate for about 24 hours until 50% to 60% of the lesions of the cells float up in a spherical shape, but the cells cannot be lysed.

[0046] 2. Purification of Whole Virus Particles of Seneca Valley Virus Hubei Strain

[0047] (1) Removal of cell debris: After cell amplification of SVV virus (cytopathic), the mixture of cells and supernatant was initially centrifuged at 4°C and 10,000 rpm for 30 min to remove cell debris.

[0048] (2) Ultracentrifuge virus ...

Embodiment 2

[0056] Preparation of monoclonal antibodies

[0057] (1) Immunization test

[0058] The immunization test used 5-week-old female BALB / c mice (purchased from the Experimental Animal Center of Huazhong Agricultural University, referred to as mice), and the whole virus particles purified and prepared in Example 1 were used as antigens. The immunization procedures are shown in Table 1:

[0059] Table 1 Mouse immunization program

[0060]

[0061] Ten days after the third immunization, blood was collected by docking the tail, and the serum titer of the mice was detected by indirect ELISA method, and the mouse with the highest serum titer was selected for subsequent experiments.

[0062] The serum titer detected by the indirect ELISA method is as follows:

[0063] 1. Establishment of indirect ELISA detection method

[0064] Determine the optimal coating concentration of the antigen and the optimal dilution factor of the serum by square array titration:

[0065] (1) Dilute the...

Embodiment 3

[0122] Bioactivity detection of monoclonal antibodies

[0123] (1) Ascites titer determination: The titer of the above-mentioned monoclonal antibody in ascites was detected by indirect ELISA method. After coating the microtiter plate with the SVV whole virus particle prepared by the present invention, the ascites was mixed from a volume ratio of 1:2 1 ×100 Ratio 1:2 16 ×100 times was used as the primary antibody, HRP-labeled goat anti-mouse IgG (purchased from Wuhan Boster Biological Co., Ltd.) was used as the secondary antibody for indirect ELISA detection, and non-immunized mouse serum was used as a negative control.

[0124] Test results: the ascites titer of the monoclonal antibody described in the present invention is 1:2 13 ×100.

[0125] (2) Expression of recombinant protein pET-28a-SVV-VP1

[0126]First, the genetic software MacVector 7.2 was used to analyze the gene sequence encoding wild-type SVV VP1 to find out its codon usage bias, and to find out the sites in ...

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Abstract

The invention relates to a Seneca valley virus VP1 protein, a coding gene, a hybridoma cell line and a monoclonal antibody and application thereof, which belongs to the technical field of viruses. TheSeneca valley virus Hubei strain VP1 protein is used as a Seneca Valley virus antigen protein in immunodetection. The invention provides a hybridoma cell line 2G6 of a monoclonal antibody capable ofsecreting the Seneca Valley virus Hubei strain VP1 protein according to claim 1, and a preservation number of the hybridoma cell line is CCTCC NO. C2017226. The monoclonal antibody is secreted from the hybridoma cell line 2G6. The invention relates to application of the monoclonal antibody in immune combined Seneca Valley virus Hubei strain VP1 protein. The invention provides application of the monoclonal antibody in a kit for preventing the Seneca Valley virus Hubei strain from infecting the cells.

Description

technical field [0001] The invention belongs to the field of virus technology, in particular to Seneca Valley virus VP1 protein, coding gene, hybridoma cell line, monoclonal antibody and application thereof. Background technique [0002] The Seneca Valley Virus (SVV) genome is a single-stranded, positive-strand, non-segmented RNA with no envelope and a particle diameter of 25-30 nm. It is a member of the small RNA virus family Senecavirus genus sole member. It can cause blisters and ulcers in the oral mucosa, nose and hooves of sows and fattening pigs, resulting in anorexia, high fever and lameness; sudden death, severe or occasional fatal diarrhea, dehydration and lethargy in piglets and other infectious blisters disease. [0003] In 2002, a genetic therapy company in the United States accidentally discovered the virus in PER.C6 cell culture, identified it as a potential oncolytic virus for tumors of neuroendocrine origin, and named it SVV-001, and it was not fully tested...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/085C12N15/41G01N33/569G01N33/577C12N5/20C07K16/10A61K39/42A61P31/14
CPCA61P31/14C07K14/005C07K16/1009C07K2317/76C12N2770/32022G01N33/56983G01N33/577G01N2333/085
Inventor 钱平莫红芳李祥敏陈鑫姚慧敏金梅林陈焕春
Owner HUAZHONG AGRI UNIV
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