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Establishment method of indirect ELISA (enzyme-linked immunosorbent assay) for detection of Brucella abortus HSP70

A technology for establishing methods for Brucella bovis, which is applied in the field of establishing indirect ELISA, and can solve problems such as low isolation rate, high sensitivity of PCR detection method, false positives, etc.

Inactive Publication Date: 2019-03-19
HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacterial isolation is the "gold standard" for the diagnosis of brucellosis, but Brucella is a dangerous pathogen with high test requirements and a very low isolation rate, which is not suitable for large-scale quarantine; PCR detection method has high sensitivity and poor specificity; Complement fixation test has good specificity and sensitivity, but also has the disadvantage of high test requirements; tiger bengal agglutination test and test tube agglutination test have high sensitivity, but they are easy to cause false positives

Method used

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  • Establishment method of indirect ELISA (enzyme-linked immunosorbent assay) for detection of Brucella abortus HSP70
  • Establishment method of indirect ELISA (enzyme-linked immunosorbent assay) for detection of Brucella abortus HSP70
  • Establishment method of indirect ELISA (enzyme-linked immunosorbent assay) for detection of Brucella abortus HSP70

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Experimental program
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Embodiment 1

[0033] The PCR amplification of embodiment 1 HSP70 gene

[0034] The gene sequence of HSP70 was obtained from the genome of Brucella.abortus 2308. According to the expression plasmid pET32a and gene sequence analysis, BamHI and XhoI were selected as restriction sites, and Primer Premier 5.0 was used to design primers.

[0035] The primer sequences are as follows:

[0036] P1: 5'-GCggatccATGGCTAAAGTTATTGGTATC-3'; BamH I; SEQ ID NO: 1;

[0037] P2: 5'-GCctcgagTTAGCGGATCTGGTGCTCCTT-3'; Xho I; SEQ ID NO:2.

[0038] Using the genomic DNA of Brucella.abortus 2308 as a template, use the above primers for PCR amplification. The PCR reaction system is:

[0039]

[0040] The PCR reaction program was 94°C for 5min; 94°C for 30s, 56°C for 30s, 72°C for 1.5min, a total of 30 cycles; and finally 72°C for 5min.

[0041] After the reaction was completed, agarose gel electrophoresis was carried out, and the PCR product was recovered with a gel recovery kit to obtain a band with a size of...

Embodiment 2

[0042] Embodiment 2 Construction of recombinant expression vector and expression strain

[0043] The HSP70 gene obtained in Example 1 and the plasmid pET32a were subjected to double enzyme digestion, recovered from the gel, ligated and transformed, coated with LB / Amp plates, and cultured upside down in an incubator. Pick the single clone on the LB / Amp plate, shake the bacteria, and use the universal primers to carry out bacterial liquid PCR identification, and obtain the positive clone pET32a-HSP70;

[0044] Among them, the general primer sequence is as follows:

[0045] S-Tag primer: 5'-CGAACGCCAGCACATGGACA-3'; SEQ ID NO: 3;

[0046] T7 terminatorprimer: 5'-TGCTAGTTATTGCTCAGCGG-3'; SEQ ID NO: 4.

[0047] The PCR reaction system is:

[0048]

[0049] The PCR reaction program was 94°C for 5min; 94°C for 30s, 52°C for 30s, 72°C for 1.5min, a total of 30 cycles; and finally 72°C for 5min.

[0050] The positive clone pET32a-HSP70 was expanded and cultivated, and the plasmid...

Embodiment 3

[0052] Induced expression of embodiment 3 fusion protein

[0053] The expression of the recombinant expression strain pET32a-HSP70 / BL21(DE3) was induced by using IPTG with a final concentration of 1mM at 37°C and 200rpm, and an empty control group of pET32a / BL21(DE3) was set at the same time; the cells were collected after 6h and boiled. SDS-PAGE analysis was carried out after the sample, after Coomassie brilliant blue staining and decolorization, the recombinant expression strain pET32a-HSP70 / BL21(DE3) was induced to obtain a fusion protein of about 70kD, and the size was in line with expectations; no protein band was obtained in the empty load, and the results were as follows figure 2 shown.

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Abstract

The invention discloses an establishment method of indirect ELISA (enzyme-linked immunosorbent assay) for detection of Brucella abortus HSP70. The establishment method includes the steps of preparinga fusion protein of Brucella abortus HSP70, and purifying the fusion protein to obtain a purified protein; using the purified protein as an antigen to detect a Brucella serum sample, and determining antigen coating concentration, serum dilution, confining liquid type, secondary antibody diluted concentration and color rendering time; determining a critical value of indirect ELISA; determining an ELISA results judging standard; analyzing sensitivity and specificity of an indirect ELISA method. HSP70 is subjected to prokaryotic expression to obtain the purified protein; the purified protein is used as the antigen to establish the indirect ELISA method for detection of Brucella abortus; the important method is provided for the serological test of brucellosis.

Description

technical field [0001] The invention relates to a method for establishing an indirect ELISA for detecting Brucella bovis HSP70, belonging to the technical field of bioengineering. Background technique [0002] Brucellosis seriously affects the development of animal husbandry and poses a great threat to human health. Usually, human infection is mainly through contact with carrier animals or consumption of animal products contaminated by bacteria. Therefore, controlling and eliminating carrier animals is of key significance for controlling the disease. The screening of animal brucellosis relies on laboratory diagnosis. The diagnostic methods recommended by the International Veterinary Office (OIE) include: isolation and identification of Brucella, tiger bengal agglutination test, test tube agglutination test, PCR technology and complement fixation test Wait. Bacterial isolation is the "gold standard" for the diagnosis of brucellosis, but Brucella is a dangerous pathogen with...

Claims

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Application Information

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IPC IPC(8): G01N33/569
CPCG01N33/56911
Inventor 吴同垒于秀剑庞洪泽李巧玲杜万年肖丽荣张志强史秋梅
Owner HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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