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

A foot-and-mouth disease virus, differential diagnosis technology, applied in the field of multi-species universal ELISA kits, kits, can solve problems such as unproven domains

Inactive Publication Date: 2016-01-13
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it has not been confirmed how the fused domai

Method used

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  • Multi-species universal ELISA kit for differential diagnosis of foot and mouth disease virus infection
  • Multi-species universal ELISA kit for differential diagnosis of foot and mouth disease virus infection
  • 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] Embodiment 1 recombinant protein [AGL] n Gene design and concatenation

[0078] 1. Materials and methods

[0079] 1.1 Materials

[0080] Recombinant plasmid pET-30a-8BF, expression vector pET-30a(+), Escherichia coli competent cells TG1 and Rosetta(DE3)PlysS were all preserved by our laboratory; cloning vector pJWF was purchased from Beijing Liuhe Huada Gene Technology Co., Ltd., The cloning vector pMD18-T was purchased from Treasure 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 preserved by our laboratory. Restriction enzymes BamHI, XhoI, BglII and T4DNA polynucleic acid kinase (T4PNK) were purchased from Treasure Bioengineering (Dalian) Co., Ltd.; T4DNA ligase was purchased from NewEnglandBioLabs (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 immun...

Embodiment 2

[0163] Embodiment 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; GelExtractionMiniKit and nitrocellulose membrane (NC membrane) were purchased from Shanghai Huashun Bioengineering Co., Ltd.; protein purification kit Ni-NTAHisHindPurificationSystem kit was purchased from Qiagen; EasySeeWesternBlotKit and WesternMarker were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0167] 1.2 Method

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

[0169] For the B-SPPAGL gene on pET-30a(+)-B-SPPAGL, the nucleotide sequences of XhoI, BglII and BamHI enzyme cleavage sites that are convenient for tandem genes are added at the beginning and the end of the design, wherein BglII and BamHI are mutually Homologous enzyme, the B-SPPAGL fragment with cohesive ends recovered after double digestion of pE...

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)-labeled antibody and goat anti-rabbit IgG horseradish peroxidase (HRP)-labeled antibody were purchased from Sigma Company; Brucella antibody rapid detection test kit and Brucella rapid detection test strips were purchased from South Korea Anjie Company; WesternMarker was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; 3ABC antibody detection ELISA kit was purchased from China Agriculture Lanzhou Veterinary Research Institute of the Academy of Sciences; HumanIgM and HumanIgA 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 Co., Ltd.

[0207] 1.2 Method

[0208] 1.2....

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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 general ELISA kit for differential diagnosis of foot-and-mouth disease virus infection, which belongs 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 profiles. Single domains of SpA, SpG and PPL have been shown to bind Ig (Jansson Birger, Uhlén Mathias, Nygren Per .AllindividualdomainsofstaphylococcalproteinAshowFabbinding[J].FEMSImmunology&MedicalMicrobiology,2006,20(1). It was previously reported that SPA can only bind to the Fc region of mouse IgG, while SPG can bind to both the Fc region and the Fab region, and it is believed that the binding sites of SPA, SPG and Fc overlap. However, Aybay, C et al. found that the binding sites of the two proteins binding to the Fc region were independent and did not over...

Claims

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Application Information

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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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