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Indirect ELISA detection method for detecting porcine epidemic diarrhea virus antibody and kit thereof

A porcine epidemic diarrhea and kit technology, applied in the direction of virus/phage, virus, virus peptide, etc., can solve the problems of high cost, the expression product cannot effectively simulate the natural conformation of viral protein, and the expression amount is low.

Pending Publication Date: 2020-07-07
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Indirect ELISAs have been developed based on whole virus preparations or recombinant viral proteins; however, because whole virus culture is time-consuming, expensive and not suitable for large-scale production; therefore, using heterologous expression systems to obtain recombinant PEDV proteins has become a research trend
[0005] At present, ELISA diagnostic kits based on PEDV M protein, N protein, S protein and ORF3 protein and PEDV whole virus have been reported; however, the preparation of protein antigens, especially S protein antigens, is mainly based on prokaryotic expression systems for expression and purification. Its expression products cannot effectively simulate the natural conformation of viral proteins, which is relatively time-consuming and labor-intensive, and the expression level is low, and the purity and specificity are low

Method used

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  • Indirect ELISA detection method for detecting porcine epidemic diarrhea virus antibody and kit thereof
  • Indirect ELISA detection method for detecting porcine epidemic diarrhea virus antibody and kit thereof
  • Indirect ELISA detection method for detecting porcine epidemic diarrhea virus antibody and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction of expression vector pPIC9K-COE and induction and identification of recombinant porcine epidemic diarrhea virus COE protein

[0059] 1. Construction of expression vector pPIC9K-COE and induced expression of recombinant porcine epidemic diarrhea virus COE protein

[0060] 1) Construction of expression vector pPIC9K-COE

[0061] The porcine epidemic diarrhea virus gene (Accession no.KP162057) was cloned into the eukaryotic expression vector pPIC9K by restriction endonucleases EcoR I and Not I, and the correct recombinant expression plasmid pPIC9K- COE was linearized, and the digested products were recovered, and electrotransfected into freshly prepared Pichia pastoris GS115 competent cells.

[0062] 2) Induced expression

[0063] The correct transformant pPIC9K-COE-GS115 plate identified by PCR was streaked on the MD plate medium, cultured at 28°C for 3 days, picked a single colony, inoculated in 5mL YPD medium, 30°C, 250r / min shaking for 24h. Th...

Embodiment 2

[0072] The establishment of the indirect enzyme-linked immunoassay (ELISA) detection method of embodiment 2 porcine epidemic diarrhea virus antibody

[0073] 1. Reagents and instruments

[0074] Skimmed milk powder: product of BD Company in the United States; HRP-labeled goat anti-pig IgG antibody: product of SIGMA Company in the United States; 96-well microplate plate: product of Shenzhen Jincanhua Company; water-proof electric heating constant temperature incubator (Shanghai Yuejin), microplate reader ( Bole) etc.

[0075] Preparation of related reagents for indirect ELISA:

[0076] 1) Coating solution: 0.05M Tris;

[0077] 2) Serum diluent and enzyme-labeled antibody diluent: PBS (0.01M, pH 7.4);

[0078] 3) Washing solution: 8g NaCl, 3g Na 2 HPO 4 12H 2 O, 0.6mL Tween-20, add distilled water to 1000mL;

[0079] 4) Blocking solution: PBS+5% skimmed milk powder;

[0080] 5) Substrate buffer A: carbamide peroxide 1g, 10.3g citric acid, 35.8g Na2HPO4·12H2O, Tween-20100...

Embodiment 3

[0136] The preparation and use of the indirect ELISA detection kit of embodiment 3 porcine epidemic diarrhea virus antibody

[0137] 1. Kit components

[0138] This embodiment is based on the indirect ELISA detection method of the porcine epidemic diarrhea virus antibody established in the above embodiment 2, and the indirect ELISA detection kit of the porcine epidemic diarrhea virus antibody is constructed, and the kit includes the following components:

[0139] (1) ELISA plate: the wells of the ELISA plate are coated with recombinant porcine epidemic diarrhea virus COE protein, and the unadsorbed sites on the surface of the micropores are sealed;

[0140] (2) Enzyme marker: horseradish peroxidase (HRP)-labeled goat anti-pig IgG antibody diluted 1:10000;

[0141] (3) Positive control serum: PEDV hyperimmune serum, diluted 1:400 with serum diluent;

[0142] (4) Negative control serum: pig serum that is sterile and not injected with any vaccine, and diluted 1:400 with serum d...

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Abstract

The invention discloses an indirect ELISA detection method for detecting a porcine epidemic diarrhea virus antibody and a kit thereof. The recombinant porcine epidemic diarrhea virus COE protein is successfully expressed by cloning a gene for encoding the COE protein into a eukaryotic expression vector pPIC9K and then transforming the eukaryotic expression vector pPIC9K into a pichia pastoris GS115 competent cell for inducible expression. Compared with a prokaryotic expression inclusion body antigen, the purification production process of the COE protein is simple, convenient and feasible, andthe COE protein is high in expression quantity and purity and is closer to a virus protein natural structure; the COE protein is used as a coating antigen; the indirect ELISA detection method for detecting the porcine epidemic diarrhea virus antibody and the kit thereof are successfully constructed, the detection results of the method and the kit thereof are high in accuracy, strong in specificity, high in sensitivity and good in repeatability, and the kit needs a small amount of samples, is simple, convenient and rapid to operate and is low in cost; therefore, the method and the kit thereofhave a wide application prospect in detection of the porcine epidemic diarrhea virus antibody.

Description

technical field [0001] The invention belongs to the technical field of animal epidemic detection. More specifically, it relates to an indirect ELISA detection method for detecting porcine epidemic diarrhea virus antibody and a kit thereof. Background technique [0002] Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is an acute infectious disease caused by coronavirus porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV). High lethality is the main feature. The disease was first reported in the United Kingdom and Belgium in the 1970s (Oldham, 1972). Subsequently, many Asian countries, including China, Japan, and South Korea, also reported the occurrence of this disease, with frequent outbreaks and serious economic consequences. loss. [0003] Porcine epidemic diarrhea virus (PEDV) is a single-stranded positive-sense RNA virus that belongs to the family Coronaviridae and the genus Coronaviridae in genetic classification. Its genome encodes fou...

Claims

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Application Information

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IPC IPC(8): G01N33/569C12N15/50C12N15/81C07K14/165
CPCC07K14/005C12N15/815C12N2770/20022G01N33/56983G01N2469/20
Inventor 王弘李莉萍杨金易肖治理苏晓娜李家冬
Owner SOUTH CHINA AGRI UNIV
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