Kit for identifying brucella abortus and other brucella species

A technology for Brucella bovis and Brucella species is applied in the field of kits for identifying Brucella bovis and other species of Brucella, which can solve the problem of interfering with the purification work of herds and inability to distinguish immunized animals. Natural virulent strains of infection antibodies and other issues

Active Publication Date: 2021-11-12
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different types of Brucella have different pathogenicity, and the types of vaccines used for immune prevention of Brucellosis are also different. In production practice, the identification of Brucella is of great significance for evaluating disease hazards and vaccine selection; and The existing commonly used diagnostic methods for brucellosis cannot distinguish the vaccine antibodies in the sera of immunized animals from the infection antibodies of natural virulent strains, which seriously interferes with the decontamination of herds. The use of bacterial vaccines is beneficial to herd decontamination

Method used

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  • Kit for identifying brucella abortus and other brucella species
  • Kit for identifying brucella abortus and other brucella species
  • Kit for identifying brucella abortus and other brucella species

Examples

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Embodiment 1

[0047] Cloning, prokaryotic expression of embodiment 1 Brucella bovis deletion gene NC_017250 and Brucella BP26 gene

[0048] Through the whole genome sequencing of the A19 vaccine strain, the predicted coding gene was compared with the published Brucella genome data to find out the missing gene of the bovine species compared with other non-bovine species of Brucella, and use the PCR method from S2 The deletion gene NC_017250 (SEQ ID NO.1) was amplified in the genome, and the Brucella BP26 gene (SEQ ID NO.3) was used as a control gene. After cloning and sequencing, it was constructed into the prokaryotic expression vector pET30 and transformed into E .coli BL21(DE3) was induced to express, and the immunogenicity of the two recombinant proteins NC_017250 and BP26 was verified by western-blotting method.

[0049] 1. Experimental method:

[0050] 1.1 Primer design

[0051] According to the NC_017250 and BP26 gene sequences, primers were designed with Oligo6.0, and EcoRI and Xho...

Embodiment 2

[0118] Example 2 Establishment of iELISA method for identifying bovine and non-bovine Brucella antibodies in domestic animals

[0119] 1. Experimental method

[0120] 1.1 Determination of the optimal coating concentration of antigen and the optimal dilution of serum

[0121] According to the checkerboard titration method, the two recombinant proteins purified in Example 1 were diluted with the coating solution. The initial antigen concentration of NC_017250 was 0.5 μg / μL, and the dilution gradient was 1:6.25, 1:12.5, 1:25, 1 : 50 four gradients; BP26 initial antigen concentration 0.553μg / μL dilution gradient is 1:25, 1:50, 1:100, 1:200 four gradients, add 100μL to each well, coat at 4°C overnight, the next day Discard the liquid in the well, wash it three times with washing solution, let it stand for 5 minutes each time, and pat dry the liquid in the well of the microplate after washing. Add 100 μL of blocking solution to each well, place at 37°C for 2 hours, wash with washi...

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Abstract

The invention provides a kit for identifying brucella abortus and other brucella species. The kit is coated with a protein as shown in SEQ ID NO. 2 and a brucella BP26 protein. The invention finds that the protein as shown in SEQ ID NO.2 or the coding gene thereof can be used for distinguishing the brucella abortus from other brucella species. The invention provides an iELISA detection reagent for identifying brucella abortus infection serum and other brucella infection serum, and the reagent contains the two proteins, or contains recombinant proteins with His tags fused at the N ends or C ends of the two proteins. The iELISA detection reagent provided by the invention can quickly identify antibodies of bovine vaccine immunization or wild viruses and other species (sheep, pigs, dogs and the like) vaccine immunization or wild viruses in livestock serum, solves the problem of difficult identification of Brucella strain types in livestock groups, and provides reference for pathogen identification, vaccine selection and livestock group purification of livestock brucellosis.

Description

technical field [0001] The invention relates to the fields of genetic engineering and immunology, in particular to a kit for distinguishing Brucella bovis from other Brucella species. Background technique [0002] Brucellosis (brucellosis) is caused by Brucella (brucella spp.) worldwide widespread zoonosis, OIE classified as B infectious diseases. Brucella is divided into six classic species, cattle (B.abortus), sheep (B.melitensis), pigs (B.suis), dogs (B.cains), sheep (B.ovis) and In recent years, B.neotomae and B.ceti and B.pinnipedialis have been found successively in humans, rodents and bats. Brucella has also been found in animals such as sheep, cattle, and pigs as the main epidemic pathogenic species of domestic animals and humans. 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 org...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/31C12Q1/689G01N33/543G01N33/569
CPCC07K14/195C12Q1/689G01N33/56911G01N33/543G01N2333/23Y02A40/70
Inventor 王文龙王姝懿陆静毛晓伟呼和巴特尔刘春霞
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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