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Hybridoma cell strain and secretion monoclonal antibody and application thereof

A technology of hybridoma cells and monoclonal antibodies, applied in the direction of antibodies, analytical materials, antiviral agents, etc., can solve the problems of high cost, re-emergence of piglets, single preventive effect of vaccines, etc., and achieve good neutralizing activity and high binding strength Effect

Active Publication Date: 2015-06-10
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the existing commercial vaccines at home and abroad are all classic vaccine strains. Although they have a certain effect on controlling the disease, they still cannot provide good passive protection for piglets.
At the same time, the level of maternal antibody obtained by piglets is not uniform, which can easily cause some piglets to get sick again and cause the pathogen to spread rapidly
Although yolk antibodies have been used to prevent or treat porcine epidemic diarrhea virus, yolk antibodies are high in protein, not only costly, but also prone to bacterial infection in the production process and difficult for large-scale production (Zhang Jiazheng, Shi Ping, Niu Guiling. Advantages, disadvantages and prospects of using high-immune yolk antibody solution. China Poultry, 2003,25(23):45)
Based on this, a new approach is urgently needed to solve the existing vaccines, the porcine epidemic diarrhea virus infection caused by insufficient maternal antibodies, and the single preventive effect of existing vaccines

Method used

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  • Hybridoma cell strain and secretion monoclonal antibody and application thereof
  • Hybridoma cell strain and secretion monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The acquisition of embodiment 1 hybridoma cell strain

[0057] 1.1 Propagation and purification of PEDV virus

[0058] 1.1.1 Antigen preparation

[0059] Wash the Vero cells that have grown into a good monolayer (the Vero cells are from Shanghai Institute of Biochemistry) for 2-3 times with PBS, and inoculate the PEDV HN1301 strain at a dose of MOI=0.001. Incubate at 37°C for 60 minutes, mix gently every 20 minutes during this period, discard the liquid in the bottle after adsorption, add 5mL of fresh maintenance solution, and place at 37°C CO 2 In the incubator, the virus liquid was harvested when the cytopathic rate reached 90%, and frozen at -20°C for later use.

[0060] 1.1.2 Antigen purification

[0061] Take 200mL of virus liquid and add 20mL TritonX-100, shake at room temperature for 10min, centrifuge at 5000rpm in a high-speed refrigerated centrifuge for 30min, take the centrifuged supernatant in an ultra-speed refrigerated centrifuge at 40000rpm, centrifuge ...

Embodiment 2

[0110] Example 2 Preparation and Identification of Monoclonal Antibody

[0111] 2.1 Preparation of monoclonal antibody ascites First, sensitize BALB / C female mice with sterilized liquid paraffin, inject 0.5 mL / peritoneally, and inject 0.5 mL (about 2.0×10 6 cells / mL) of hybridoma cells. Observation, ascites was taken when the abdomen of the mouse was obviously enlarged. Put the ascitic fluid at 37°C, and then overnight at 4°C after 1 hour. The supernatant was collected by centrifugation the next day, aliquoted, and stored at -70°C.

[0112] 2.2 Identification of monoclonal antibody Ig type

[0113] The Ig type of monoclonal antibody was identified by using the monoclonal antibody typing reagent of Sigma Company according to the instructions. The heavy chain of 8A3A10 monoclonal antibody was IgG2a subtype, and the light chain was Kappa chain.

[0114] 2.3 Determination of monoclonal antibody titer

[0115] Using the IFA method in Example 1.4.2, the IFA titer of the prepare...

Embodiment 3

[0134] Example 3 Monoclonal Antibody Prevention, Effect Evaluation of Treatment Virus Infection

[0135] 3.1 Evaluation of the effect of monoclonal antibody in preventing viral infection

[0136] Screen PEDV, RV, TGEV antigen, 15 2-3 day-old piglets of double-negative age of antibody, be divided into 3 groups at random, the monoclonal antibody 2mL that the first group and the second group are prepared in oral embodiment 2 respectively, the third group orally 2 mL of PBS buffer, 24 hours after oral administration, the first group and the third group were orally administered 2 mL of PEDV epidemic strain HN1301 small intestinal virus (containing 1000 minimum incidence doses), and the second group were orally administered CV777 small enterotoxin (containing 1000 minimum incidence doses), The clinical symptoms of piglets were observed daily for 14 consecutive days, and the effect of the monoclonal antibody on preventing diarrhea virus infection was evaluated by the clinical morbidi...

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Abstract

The invention discloses a hybridoma cell strain and an anti-porcine-epidemic-diarrhea-virus monoclonal antibody produced by the hybridoma cell strain. The anti-porcine-epidemic-diarrhea-virus monoclonal antibody has the preventing and / or treating function for porcine epidemic diarrhea viruses, solves the problem of piglet epidemic diarrhea virus infection caused when existing vaccines and maternal antibodies are insufficient, overcomes the defect that an existing vaccine is single in prevention function and can also be used for developing diagnostic reagent products for the porcine epidemic diarrhea viruses.

Description

technical field [0001] The invention relates to a hybridoma cell strain, a monoclonal antibody secreted by it, a pharmaceutical composition prepared by using the secreted monoclonal antibody, a test kit and an application, and belongs to the field of biomedicine. Background technique [0002] Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is an acute, contact and highly contagious digestive tract disease caused by porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), the main symptoms are vomiting, watery diarrhea and dehydration , The mortality rate of piglets within 7 days of infection is as high as 80%-100%. Cause huge loss to my country's pig industry. Since 2010, there have been large-scale outbreaks of diarrheal diseases in autumn and winter in my country. At the same time, large-scale outbreaks of diarrheal diseases have also occurred in South Korea, Japan, Taiwan, the United States and other places. Molecular biology studies have show...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/10A61K39/42A61P31/14G01N33/577G01N33/569
Inventor 田克恭王延辉张云静
Owner LUOYANG PULIKE WANTAI BIOTECH
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