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A Genetic Engineering Antigen and Antibody of Porcine Epidemic Diarrhea Virus

A technology for porcine epidemic diarrhea and genetically engineered antibodies, applied in genetic engineering, viral peptides, plant genetic improvement, etc., can solve problems such as poor immune effect, high cost, and low IgY yield

Active Publication Date: 2020-03-27
HENGYANG NORMAL UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some IgY preparations in the experimental stage are mostly prepared by traditional whole-virus inactivated vaccines to prepare immunogens for immunization by extracting laying hens. This method is costly, the purity of antigens is not high, and the effective antigen content cannot be guaranteed, which eventually leads to immune effects. Poor, low IgY output, eventually leading to outstanding problems such as poor control effect

Method used

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  • A Genetic Engineering Antigen and Antibody of Porcine Epidemic Diarrhea Virus
  • A Genetic Engineering Antigen and Antibody of Porcine Epidemic Diarrhea Virus
  • A Genetic Engineering Antigen and Antibody of Porcine Epidemic Diarrhea Virus

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Experimental program
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preparation example Construction

[0075] Concrete preparation method of the present invention comprises:

[0076] 1. PEDV culture, RNA extraction and first-strand cDNA synthesis

[0077] 1.1 Vero cells of African green monkey kidney cells, using DMEM medium containing 10% fetal bovine serum by volume fraction, at 37 ° C, 5% CO 2 cultured in an incubator.

[0078] 1.2 When Vero cells grow to about 80% density, wash with pH7.4 phosphate buffered saline (PBS), add serum-free MEM medium, supplement trypsin to a final mass concentration of 6 μg / mL, shake well, Treat for 30min.

[0079] 1.3 Inoculate Vero cells with porcine epidemic diarrhea mutant strain PEDV-HuN14 or classic strain PEDV-C14 at 5MOI, shake gently, and place at 37°C with a volume fraction of 5% CO 2 Cultivate in an incubator for 24 to 72 hours.

[0080] 1.4 When 80% of the cells appear cytopathic (CPE), freeze and thaw repeatedly 3 times, so that the cells are lysed to release virus particles.

[0081] 1.5 Use Trizol reagent to extract total RN...

Embodiment 1

[0152] Example 1 Immune Activity Identification of Porcine Epidemic Diarrhea Virus Genetic Engineering Bivalent Egg Yolk Antibody Preparation

[0153] The above-mentioned polyclonal antibody prepared by the present invention was initially diluted 1:2000 times, and on this basis, it was diluted by 2 times until 1:64 000 times, and the titer of the polyclonal antibody was determined by Western blot technique. Such as Figure 13 As shown, the IgY antibody titer reached 1:16 000 times.

Embodiment 2

[0154] Example 2 Evaluation of Reactivity and Neutralization Titer of Porcine Epidemic Diarrhea Virus Genetic Engineering Bivalent Egg Yolk Antibody Preparation to Virus

[0155] Cultivate Vero cells and inoculate porcine epidemic diarrhea mutant strain PEDV-HuN14 or classic strain PEDV-C14 when they grow to 60%-80% confluence, discard the medium after 48 hours of infection, and use a volume fraction of 33% Fix with acetone-PBS for 30 min, add polyclonal antibody (diluted 1:100) after drying, set up non-immune negative serum as a control group, incubate at 37°C for 1 h, wash with PBS 3 times, add HRP-SPA (diluted 1:3000), 37 Incubate at ℃ for 1 hour, wash with PBS 3 times, add AEC chromogenic solution, incubate at 37 ℃ for 30 minutes, discard the chromogenic solution and add deionized water to terminate the reaction, observe and judge the result under an inverted microscope.

[0156] Such as Figure 14As shown, the porcine epidemic diarrhea virus genetically engineered bivale...

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Abstract

The invention discloses a porcine epizootic diarrhea virus gene engineering antigen and antibody. A classical strain and a variant strain of a virus are used as base materials, a test verifies that the sequence of an original genetic gene is systematically transformed and optimized and is subjected to manual de novo synthesis to separately obtain three protective antigen genes sourced from the classical strain and three protective antigen genes sourced from the variant strain, the six genes are separately connected with a prokaryotic expression vector, and a recombinant expression strain is obtained after screening and identification; a target protein is obtained through IPTG induction; and six proteins are purified, and are mixed at the mass ratio of 1:1:(2-4):1:1:(2-4), and white oil orother immunopotentiators is or are added to prepare a water-in-oil type immunogen. Laying hens are immunized, eggs are collected, and an egg yolk antibody is extracted from egg yolk. An antibody preparation is orally taken by newborn suckling piglets, so that the infection of the porcine epizootic diarrhea classical strain and the porcine epizootic diarrhea variant strain can be prevented, the clinical application range is greatly expanded, and the treatment and prevention effects are improved.

Description

technical field [0001] The invention belongs to the technical field of preparing antigens and antibodies by genetic engineering, and in particular relates to a main protective antigen and yolk antibody of porcine classical strains and variant strains and a preparation method thereof. Background technique [0002] Porcine epidemic diarrhea (porcine epidemic diarrhea, PED) is an intestinal infectious disease caused by porcine epidemic diarrhea virus (porcine epidemic diarrhea virus, PEDV), characterized by watery vomiting, diarrhea, and dehydration, with a mortality rate of up to 100%. . Infection by classic PEDV strains has a slow transmission speed and is mainly characterized by endemicity. Since 2010, new PEDV variant epidemic strains have emerged in my country, highlighting the characteristics of rapid transmission, high morbidity and high mortality. Since April 2013, the PED epidemic broke out in North America and spread rapidly, causing huge economic losses to the pig i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/165C07K16/10C07K16/02C12N15/50
CPCC07K14/005C07K16/02C07K16/10C07K2317/11
Inventor 唐青海孙国印魏平华杨海李晓楠张抒王瑞峰曹书显唐斯萍杨灿王芳宇
Owner HENGYANG NORMAL UNIV