ELISA detection kit for bovine Brucella

A technology of Brucella bovis and detection kit, which is applied in the field of protein engineering to achieve the effects of easy operation, high accuracy and low cost of use

Active Publication Date: 2016-03-30
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no research report on mixing OMP19,

Method used

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  • ELISA detection kit for bovine Brucella
  • ELISA detection kit for bovine Brucella
  • ELISA detection kit for bovine Brucella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of recombinant expression vector of Brucella bovis outer membrane protein

[0037] 1. Primer design and synthesis

[0038] According to the Brucella bovis outer membrane protein gene sequence OMP19 (accession number L27997), OMP22 (accession number AY484562), OMP28 (accession number JQ865997) published on GenBank, the upstream and downstream primers were designed using primer5.0 software. , Send the designed primers to Shanghai Shenggong Biological Engineering Technology Co., Ltd. for synthesis. The primer sequence is as follows:

[0039] F omp19 5’-cgc ggatcc ATGGGAATTTCAAAAGCAAG-3’,

[0040] R omp19 5’-ccg ctcgag TCAGCGCGACAGCGTCAC-3’;

[0041] F omp22 5’-cgc ggatcc ATGTTCAAGCGTTCTATC-3’,

[0042] R omp22 5’-ccg ctcgag CTAGAATTTGTAGTTCAG-3’;

[0043] F omp28 5’-cgc ggatcc ATGAACACTCGTGCTAGC-3’,

[0044] R omp28 5’-ccg ctcgag TTACTTGATTTCAAAAACG-3’.

[0045] Underlined are BamHI and XhoI restriction sites respectively.

[0046] 2. Amplification of t...

Embodiment 2

[0052] Example 2 Induced expression and purification of recombinant protein from the outer membrane of Brucella bovis

[0053] 1. Induction and expression of recombinant protein

[0054] The sequencing gene of Example 1 and the recombinant plasmid pCold-TF / OMP19, pCold-TF / OMP22, and pCold-TF / OMP28 with the correct reading frame were transformed into E.coli BL21(DE3) competent cells, respectively. Cultured on LB medium with 100μg / ml ampicillin. The bacterial solution containing the recombinant plasmid was inoculated into LB medium containing 100μg / ml ampicillin at the amount of 1%, and cultured with shaking at 37°C for about 2h, to OD 600 The value is 0.6-0.8, the inducer IPTG is added to the final concentration of 0.5mmol / L, and the bacteria are harvested after 24 hours of culture at 16°C. The bacterial solution was centrifuged at 10000g for 15min at 4℃, and then the pellet was resuspended in PBS buffer; ultrasonicated for 15min in an ice bath until the liquid became clear, and th...

Embodiment 3

[0058] Example 3 Establishment of ELISA detection method for Brucella bovis

[0059] 1. Establishment of indirect ELISA method

[0060] Use NaHCO 3 (pH=9.6) The purified recombinant proteins rOMP19, rOMP22, and rOMP28 are diluted with buffer solution, and the concentrations are respectively diluted to 1 μg / ml, and then the recombinant antigens are mixed in a 1:1:1 volume ratio as the coating antigen, and each well is added Sample 100μl, coated with 96-well ELISA plate at 37°C for 2h. Each well was blocked with 200 μl volume of 10% horse serum for 45 minutes, and washed with PBST five times. Negative and positive sera (China National Medical Products Administration) were diluted 1:50, 1:100, 1:200, 1:400 respectively, 100μl was added to each well, incubated at 37°C for 30min, washed with PBST five times. Enzyme-labeled secondary antibody (2mg / ml) was diluted with PBST 1:10000, 1:20000, 1:40,000, 100μl was added to each well, incubated at 37°C for 30min, and washed with PBST five t...

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Abstract

The invention provides an ELISA detection kit for bovine Brucella, belonging to the technical field of protein engineering. In the kit provided by the invention, the mixed antigen of the outer membrane proteins OMP19, OMP22 and OMP28 of bovine Brucella is used as an envelope antigen of the ELISA kit; the concentration of each antigen is 1mu g/ml, and the antigens are mixed in a volume ratio of 1:1:1 to obtain the mixed antigen; the serum dilution is 1:200, and the dilution of the enzyme-labeled secondary antibody is 1:20,000; and the bovine Brucella is detected through an indirect ELISA method. The kit provided by the invention has the characteristics of high specificity, high sensitivity, high accuracy, easiness in operation and good reproducibility while the market application prospect is good.

Description

Technical field [0001] The present invention relates to the technical field of protein engineering, in particular, to a Brucella bovis ELISA detection kit using Brucella bovis outer membrane protein as a detection antigen. Background technique [0002] Brucellosis is a zoonotic disease that affects severely worldwide. Brucellosis is caused by Brucella. It mainly affects the reproductive system of animals, causes miscarriage and infertility, and brings serious economic losses. The diagnostic method for confirming brucellosis is the isolation, culture and identification of pathogenic microorganisms. [0003] At present, most of the serological diagnostic methods are based on the detection of antibodies against Brucella lipoprotein (lipopolysaccharide, LPS). Although the tiger red plate agglutination and complement fixation test is the most recognized and accepted test in the world, Because the experimental results are cross-cutting, the experimental operation steps are cumbersome a...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531
CPCG01N33/531G01N33/56911G01N33/68G01N2333/23
Inventor 韩雪清王慧煜梅琳牙侯勋
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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