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Anti-porcine epidemic diarrhea virus monoclonal antibody and its cell line and application

A porcine epidemic diarrhea and monoclonal antibody technology, applied in antiviral immunoglobulin, chemical instruments and methods, and microbial-based methods, can solve the problems of rapid spread of the epidemic and heavy losses, and achieve good detection specificity and sensitivity , good linear detection range, good promotion and application value effect

Active Publication Date: 2019-04-30
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And finally caused an outbreak in Canada and Mexico. The epidemic spread rapidly and caused heavy losses.

Method used

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  • Anti-porcine epidemic diarrhea virus monoclonal antibody and its cell line and application
  • Anti-porcine epidemic diarrhea virus monoclonal antibody and its cell line and application
  • Anti-porcine epidemic diarrhea virus monoclonal antibody and its cell line and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The purification of embodiment 1 porcine epidemic diarrhea virus

[0028] 1) The PEDV cell culture was centrifuged at 3000 rpm at 4°C for 30 min, and the supernatant was collected.

[0029] 2) Ammonium sulfate precipitation Add ammonium sulfate according to the amount of 42.5g ammonium sulfate in 100ml virus liquid, stir well, put it in a refrigerator at 4°C to stir and precipitate overnight, centrifuge at 5000rpm at 4°C for 40min, remove the supernatant, and use appropriate amount of sterile ddH for precipitation 2 O suspension.

[0030] 3) Dialysis: Put the suspended virus liquid in a dialysis bag, stir and dialyze in PBS, change the liquid every half an hour, change 5 times in total, and dialyze overnight for the 5th time.

[0031] 4) Concentration: After dialysis, take out the virus and concentrate the virus to an appropriate volume with polyethylene glycol.

[0032] 5) Ultra-centrifugal purification: Add different concentrations of sucrose to the bottom of the ce...

Embodiment 2

[0033] Embodiment 2 The establishment of monoclonal antibody hybridoma cell line and the preparation of monoclonal antibody

[0034] After immunizing BALB / c mice with the PEDV virus purified in the above-mentioned Example 1, the splenocytes thereof were fused with SP2 / 0 myeloma cells. Hybridoma cell supernatants were screened for specific antibodies by indirect ELISA. The hybridoma cell line PEDV-J11E was obtained, which was deposited in China Center for Type Culture Collection on July 29, 2016, with the preservation number CCTCC NO:C2016145. It can stably cultivate and secrete monoclonal antibody against porcine PEDV. The Ig type of the obtained monoclonal antibody is IgG1,k, and the molecular weights of its heavy chain and light chain are 55KDa and 25KDa respectively; wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 2 The nucleotide sequence of the heavy chain is shown in SEQ ...

Embodiment 3

[0091] The preparation of embodiment 3 polyclonal antibody

[0092] (1) Preparation method

[0093] Select two 2Kg male New Zealand white rabbits, use Freund's complete adjuvant to emulsify and purify PEDV for initial immunization, the antigen amount is 0.5mg / Kg body weight, and inject subcutaneously at multiple points; Half of the antigen was injected subcutaneously at multiple points; after 28 days, the third immunization was carried out according to the dose of the second immunization; after 28 days, the booster immunization was carried out according to the dose of the second immunization; blood was collected from the heart 7 days later, the serum was separated, and frozen at -20°C for later use.

[0094] (2) Determination of titer of multiple antiserum

[0095] The method is the same as that of monoclonal antibody. Serum titers before and after immunization of rabbits showed that the polyantibody titer after immunization was 1:64000.

[0096] (3) Determination of the spec...

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Abstract

The invention discloses a porcine epidemic diarrhea virus monoclonal antibody, a cell strain thereof and an application. The cell strain PEDV-J11E is collected at the China Center for Type Culture Collection, the collection number of the cell strain is CCTCC NO: C2016145, the monoclonal antibody secreted by the cell strain has high specificity and affinity for PEDV, can be specifically combined with different epitope of the PEDV and can be used for building a double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for quantitative detection of the PEDV. The double-antibody sandwich ELISA method has excellent detection specificity, sensitivity and linear detection range, and a double-antibody sandwich ELISA kit has excellent popularization and application values.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a monoclonal antibody against porcine epidemic diarrhea virus, a cell line thereof and applications thereof. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is an acute and highly contagious intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV), characterized by severe enteritis, vomiting and watery Diarrhea is the main feature, and the clinical changes and symptoms are very similar to porcine transmissible gastroenteritis (TGE). The transmission route of PEDV is mainly horizontal transmission, and the main route is the digestive tract. Piglets will develop the disease after eating or contacting infected feed, feces, and drinking water. Low temperature, humidity, and poor sanitation will accelerate the spread of the disease. PED is an explosive epidemic. After an outbreak in most...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10G01N33/569G01N33/577C12N5/20C12R1/91
CPCC07K16/10C07K2317/51C07K2317/515
Inventor 贾爱卿王贵平刘琪周如月甘振磊
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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