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Livestock S2 vaccine immunity and Brucella melitensis/abortus infection IELISA (enzyme linked immunosorbent assay) detection reagent

A technology of Brucella bovis and detection reagent, which is applied in the fields of genetic engineering and immunology, can solve the problem of inability to identify and immunize herds to purify naturally infected animals with Brucella, and it is impossible to distinguish between immunization and immunization of Brucella vaccine. Natural infection and other problems, to achieve high specificity, sensitivity, and good repeatability

Active Publication Date: 2016-06-22
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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Problems solved by technology

Traditional vaccines are difficult to achieve the ideal level of protective immunity, and current diagnostic methods cannot distinguish Brucella vaccine immunity from natural infection, and some differential diagnosis methods studied in recent years cannot distinguish S2 vaccine immunity from S2 vaccine immunity in production practice. Brucella infection in ovine / bovine species, therefore, it is difficult to decontaminate naturally infected animals with Brucella in vaccinated herds

Method used

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  • Livestock S2 vaccine immunity and Brucella melitensis/abortus infection IELISA (enzyme linked immunosorbent assay) detection reagent
  • Livestock S2 vaccine immunity and Brucella melitensis/abortus infection IELISA (enzyme linked immunosorbent assay) detection reagent
  • Livestock S2 vaccine immunity and Brucella melitensis/abortus infection IELISA (enzyme linked immunosorbent assay) detection reagent

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Experimental program
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Embodiment 1

[0040] Cloning, prokaryotic expression of embodiment 1S2 vaccine strain gene GL_0002189 and Brucella BP26 gene

[0041] Through the whole genome sequencing of the S2 vaccine strain, the predicted coding gene is compared with the published Brucella genome data to find out the S2-specific genes compared with the sheep and bovine Brucella, and use the PCR method to extract the S2 genome The specific gene GL_0002189 (SeqIDNo.2) of the S2 vaccine strain was amplified in the vaccine strain, and the Brucella BP26 gene (SeqIDNo.3) was used as the control gene. After cloning and sequencing, it was constructed into the prokaryotic expression vector pET30 and transformed into E. coliBL21(DE3) was induced to express, and the immunogenicity of the two recombinant proteins GL_0002189 and BP26 was verified by western-blotting method.

[0042] 1.1 Experimental method:

[0043] 1.1.1 Primer design

[0044] According to the GL_0002189 and BP26 gene sequences, Oligo6.0 was used to design prime...

Embodiment 2I

[0120] Embodiment 2IELISA distinguishes the method that domestic animal S2 vaccine immunity and sheep / bovine breed Brucella infect

[0121] 2.1 Experimental method

[0122] 2.1.1 Determination of the optimal coating concentration of antigen and the optimal dilution of serum

[0123] According to the checkerboard titration method, the two recombinant proteins purified in Example 1 were diluted with the coating solution. The dilution gradients were 1:25, 1:50, 1:100, and 1:200. The initial concentrations of the antigens were respectively For GL_0002189 (0.421mg / mL) and BP26 (0.553mg / ml), add 100μl to each well, coat overnight at 4°C, discard the liquid in the well the next day, wash with washing solution three times, let stand for 5min each time, wash Pat dry the liquid in the wells of the microtiter plate after finishing. Add 100 μl of blocking solution to each well, place at 37°C for 2 hours, wash with washing solution three times, let stand for 5 minutes each time, and pat ...

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Abstract

The invention provides a livestock S2 vaccine immunity and Brucella melitensis / abortus infection IELISA (enzyme linked immunosorbent assay) detection reagent.The active ingredients of the IELISA detection reagent are a Brucella S2 vaccine strain gene GL_0002189 coded antigen protein and a Brucella BP26 protein, or recombinant proteins with N terminals or C terminals fused with His labels.The livestock S2 vaccine immunity and Brucella melitensis / abortus infection IELISA detection reagent is capable of identifying main epidemic strains-Brucella melitensis / abortus and a mainstream vaccine-S2 vaccine generally used in the market, thus, vaccine immunity in livestock serums and natural Brucella melitensis / abortus infection antibodies can be identified within short time, and the problem of brucellosis detection and herd purification interfered by vaccine antibodies in immune herds is solved.The livestock S2 vaccine immunity and Brucella melitensis / abortus infection IELISA detection reagent can bring social and economic benefits and make great contributions to purifying brucellosis in the herds.

Description

technical field [0001] The invention relates to the fields of genetic engineering and immunology, in particular to an IELISA detection reagent for livestock S2 vaccine immunization and ovine / bovine Brucella infection. Background technique [0002] Brucellosis (brucellosis) is caused by Brucella (brucella) worldwide widespread zoonosis, OIE classified as B infectious diseases. Brucella is divided into seven species, cattle (B.abortus), sheep (B.melitensis), pigs (B.suis), dogs (B.cains), sheep (B.ovis), forest Brucella rat species (B.neotomae), marine mammal Brucella species (B.meris), sheep species, and cattle species are the main epidemic pathogen species, among which the sheep species is the most virulent and the most harmful. Brucellosis is transmitted through contact with sick animals, unsterilized meat products, and dairy products. It can also invade the body through the respiratory tract, digestive tract, skin and mucous membranes, and other tissues and organs to caus...

Claims

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Application Information

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IPC IPC(8): C07K14/23C12N15/31C12N15/11G01N33/68G01N33/569
CPCC07K14/23G01N33/56911G01N33/68G01N2333/23
Inventor 王文龙红梅呼和巴特尔刘春霞刘宁
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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