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A kind of gene expression product blsj-3 of Brucella diagnostic marker function and preparation method thereof

A technology for expressing products and Brucella, applied in chemical instruments and methods, biological material analysis, instruments, etc., can solve the problem of inability to distinguish Brucella vaccine immune antibodies

Active Publication Date: 2020-05-19
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to solve the difficult problem that existing serological methods cannot distinguish Brucella vaccine immune antibodies and natural infection antibodies, provide Brucella gene expression products with differential diagnosis value, and use immunological methods to realize vaccine immune antibodies Differential diagnosis of antibodies against natural infection

Method used

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  • A kind of gene expression product blsj-3 of Brucella diagnostic marker function and preparation method thereof
  • A kind of gene expression product blsj-3 of Brucella diagnostic marker function and preparation method thereof
  • A kind of gene expression product blsj-3 of Brucella diagnostic marker function and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] ——Screening research of differential diagnosis antigen protein spot

[0050] Differential diagnostic antigens were screened from membrane proteins of S2 strain by immunoproteomics method. Each of 30 brucellosis-negative healthy sheep, 30 S2 immunized sheep, and 30 clinical brucellosis-infected sheep-positive mixed sera were used for western-blotting (Western-blotting) with S2 strain membrane protein two-dimensional electrophoresis gel (2D), respectively. Looking for the difference between the S2 membrane protein and the serum reaction of sheep with different brucellosis status (infected / immune / negative), a total of 113 differential protein spots were found. A total of 30 protein spots with large differences in immune response were selected for matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry identification and bioinformatics analysis. A total of 14 proteins were identified. These proteins undertake 13 types of molecular functions ...

Embodiment 2

[0075] ——Determination of gene information of differential diagnosis antigen protein spot

[0076] The candidate protein spots obtained by the two methods were compared with the expressed proteins in the S2 genome information published on the website of the National Center for Biotechnology Information (NCBI), and the corresponding gene information of each candidate protein spot was obtained in Table 3 (including gene name information, Gene number, gene length, protein molecular weight, etc.), then respectively design primers and use PCR for gene cloning to obtain prokaryotic expression products (Table 3, image 3 , Figure 4 ), respectively with Brucella immune serum and Brucella natural infection serum by Western-blot (Western-blot) and indirect enzyme-linked immunosorbent assay (ELISA) to verify its differential diagnosis value ( Figure 5 ,Table 4).

[0077] 1. Eight candidate differential diagnosis antigens (Table 3), numbered #1-#8 respectively, and protein numbers are...

Embodiment 3

[0093] ——8# (hypothetical protein / 31kDa outer membrane immunogenic protein, hypothetical protein / 31kDaouter-membrane immunogenic protein) differential diagnosis effect verification

[0094] 1. Utilize glutathione S-transferase (GST) affinity chromatography column and glutathione gradient elution method to purify recombinant protein 8#, obtain the purified target protein of purity more than 90% ( Figure 6 ).

[0095] 2. The results of the purified 8# recombinant protein brucellosis antibody enzyme-linked immunosorbent assay (ELISA)

[0096] Purified 8# recombinant protein under conventional conditions: optional 3 single parts of negative / immune / infected sera and 1 part of mixed negative / immune / infected sera, respectively purified recombinant protein with pH9.6 carbonate buffer The protein antigen was diluted to 100ng / mL, coated overnight at 4°C, blocked with 10% skimmed milk for 2 hours as a blocking solution, diluted 1:50 with PBS, and 1:50 for the enzyme-labeled secondary a...

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Abstract

The invention relates to a gene expression product BLSJ-3 with Brucella diagnosis and identification effect, and a preparation method thereof. According to the preparation method, the gene expression product is obtained via gene prokaryotic expression of Brucella S2 strain with GI of 490819668, and via glutathione S transferase (GST) affinity chromatographic column and glutathione gradient elution purifying. The gene expression product can be taken as a coating antigen, and as a diagnosis antigen of animal Brucella vaccine immunity and naturally infected serum detection. When the BLSJ-3 antigen is subjected to western blot with vaccine immune antibodies and natural infection antibodies, the difference is obvious. The gene expression product BLSJ-3 is taken as an indirect enzyme-linked immune adsorption assay (ELISA) coating antigen to detect hundreds of brucellosis clinical serum samples, and differential diagnosis effect is excellent.

Description

[0001] Technical field The present invention relates to a gene expression product BLSJ-3 with the function of diagnostic marker of Brucella and its preparation method, belonging to the field of veterinary microbiology diagnosis. Background technique [0002] Brucellosis (abbreviated as brucellosis) is a zoonotic infectious disease caused by Brucella. Human infection with brucellosis mainly comes from sick animals and their products. The source of infection related to human brucellosis in my country is mainly sick sheep and cattle, but Brucella melis is the most serious (Zhu Liangquan et al. Microbiology Bulletin, 2015(01): 171~177; Wu Qingmin. Veterinary Director Journal, 2011(09):46~47). [0003] Serological methods are currently the main method for diagnosing animal brucellosis, and vaccination is an important means of preventing animal brucellosis. The existing serological diagnosis of brucellosis is mainly based on the detection of anti-brucella lipopolysaccharide antibod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/23G01N33/569
CPCC07K14/23G01N33/56911G01N2333/23
Inventor 朱良全丁家波张磊蒋卉彭小薇冯宇范学政
Owner CHINA INST OF VETERINARY DRUG CONTROL
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