A multi-species universal ELISA kit for differential diagnosis of foot-and-mouth disease virus infection

A technology for differential diagnosis of foot-and-mouth disease virus, applied to measuring devices, instruments, scientific instruments, etc., can solve problems such as unconfirmed domains

Inactive Publication Date: 2017-11-07
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it has not been confirmed how the fused domains function, whether they are synergistic or inhibitory

Method used

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  • A multi-species universal ELISA kit for differential diagnosis of foot-and-mouth disease virus infection
  • A multi-species universal ELISA kit for differential diagnosis of foot-and-mouth disease virus infection
  • A multi-species universal ELISA kit for differential diagnosis of foot-and-mouth disease virus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Recombinant protein [AGL] n Gene design and concatenation

[0078] 1. Materials and methods

[0079] 1.1 Materials

[0080] The recombinant plasmid pET-30a-8BF, the expression vector pET-30a(+), E. coli competent cells TG1 and Rosetta(DE3) PlysS were all preserved in our laboratory; the cloning vector pJWF was purchased from Beijing Liuhe Huada Gene Technology Co., Ltd. The cloning vector pMD18-T was purchased from Bao Bioengineering (Dalian) Co., Ltd.; the target protein expression strain BL21-pET-30a-8BF and the empty vector control strain BL21-pET-30a were prepared and stored in our laboratory. Restriction endonucleases BamH I, XhoI, BglII and T4 DNA polynucleic acid kinase (T4 PNK) were purchased from Bao Bioengineering (Dalian) Co., Ltd.; T4 DNA ligase was purchased from New England BioLabs (NEB).

[0081] 1.2 Experimental method

[0082] 1.2.1 Design of oligonucleotide chains for the synthesis of B-SPA, B-SPG and B-PPL genes

[0083] The five immuno...

Embodiment 2

[0163] Example 2 Recombinant protein [AGL] n Prokaryotic expression and identification of

[0164] 1. Materials and methods

[0165] 1.1 Materials

[0166] Ampicillin (Amp) and kanamycin (Kan) were purchased from Sigma; GelExtraction Mini Kit and nitrocellulose membrane (NC membrane) were purchased from Shanghai Huashun Bioengineering Co., Ltd.; protein purification Kit Ni-NTA His Hind Purification System kit was purchased from Qiagen Company; EasySee WesternBlot Kit and Western Marker were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0167] 1.2 Methods

[0168] 1.2.1 B-SPPAGL gene tandem vector pET-30a(+)-B-SPP[AGL] n build

[0169] The B-SPPAGL gene on pET-30a(+)-B-SPPAGL was designed by adding nucleotide sequences of XhoI, BglII and BamHI enzyme cleavage sites to facilitate the tandem gene at both ends, where BglII and BamHI are each other Homocaudal enzyme, the B-SPPAGL fragment with sticky ends recovered after the pET-30a(+)-B-SPPAGL sequence was doub...

Embodiment 3

[0203] Example 3 Analysis and application of enzyme-labeled protein binding activity to various animal Ig

[0204] 1. Materials and methods

[0205] 1.1 Materials

[0206] 3, 3, 5, 5-tetramethylbenzidine (TMB), rabbit anti-bovine IgG horseradish peroxidase (HRP)-conjugated antibody and goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody were purchased from Sigma company; Brucella antibody rapid detection test kit and Brucella rapid detection test strip were purchased from South Korea's Anjie; Western Marker was purchased from Beijing Quanzhijin Biotechnology Co., Ltd.; 3ABC antibody detection ELISA kit was purchased from China Lanzhou Veterinary Research Institute, Academy of Agricultural Sciences; Human IgM and Human IgA were purchased from Beijing Bosheng Jingwei Technology Co., Ltd.; goat IgG, pig IgG, guinea pig IgG, human IgG, rabbit IgG, and mouse IgG were purchased from Wanhuashi (Beijing) Biotechnology Ltd.

[0207] 1.2 Methods

[0208] 1.2.1 HRP ...

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PUM

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Abstract

The invention discloses a multi-species universal AGL-ELISA kit for differential diagnosis of foot and mouth disease virus infection. The multi-species universal AGL-ELISA kit comprises an ELISA plate coated with 8BF protein and recombinant protein [AGL]n undergoing enzyme labeling. The recombinant protein [AGL]n is a multi-copy recombinant protein formed by performing gene optimization and series connection on three protein structural domains which are a B structural domain in a staphylococcus aureus A protein combination structural domain, a C2 structural domain in a streptococcus protein G protein immune globulin combination structural domain and a B3 structural domain in a peptostreptococcus magnus protein L protein immune globulin combination structural domain, wherein n is 1, 2 or 3. The recombinant protein [AGL]n undergoes enzyme labeling, an enzyme labeling product can be combined with multi-species mammal immune globulin through western-blot and ELISA detection, can serve as a universal antibody for serologic detection of various mammals, and can replace species specific antibodies to perform multi-species anthropozoonosis detection. According to an AGL-ELISA, various animal foot-and-mouth disease infections can be detected, vaccine immunity, naturally injected animals and persistently infected animals can be identified.

Description

technical field [0001] The invention relates to a kit, in particular to a multi-species universal ELISA kit for differential diagnosis of foot-and-mouth disease virus infection, belonging to the field of biotechnology. Background technique [0002] SPA, SPG, and PPL are typical immunoglobulin-binding proteins that can bind to human and various animal immunoglobulins with different binding abilities and binding spectra. Single domains of SpA, SpG and PPL have been shown to bind Ig (Jansson Birger, Uhlén Mathias, Nygren Per . All individual domains of staphylococcal protein A show Fab binding [J]. FEMS Immunology & Medical Microbiology, 2006, 20(1). It was previously reported that SPA can only bind to the Fc region of murine IgG, and that SPG can bind to both the Fc region and the Fab region, and that the binding sites of SPA, SPG and Fc overlap. However, Aybay, C et al. found that the binding sites of the two proteins were independent and did not overlap after digesting mo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/531
CPCG01N33/531G01N33/56983
Inventor 王君伟高明春彭统全李迪马波
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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