Indirect ELISA method of clostridium perfringens beta2 toxin antibody

A technology for Clostridium perfringens and β2 toxins, which can be used in measurement devices, instruments, scientific instruments, etc., can solve the problems of lack of detection products for toxins, and achieve the effects of high sensitivity, strong specificity and high sensitivity

Pending Publication Date: 2021-11-26
NINGXIA UNIVERSITY
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Problems solved by technology

[0005] At present, there is still a lack of relevant detection products for the detection of this toxin, and there is an urgent need for an easy-to-operate, high-sensitivity, specificity, stable and reliable method for the epidemiological investigation of Clostridium perfringens in farm animals Therefore, it is very necessary to establish an indirect ELISA detection method for the monoclonal antibody of Clostridium perfringens β2 toxin

Method used

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  • Indirect ELISA method of clostridium perfringens beta2 toxin antibody
  • Indirect ELISA method of clostridium perfringens beta2 toxin antibody
  • Indirect ELISA method of clostridium perfringens beta2 toxin antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1: Expression of soluble recombinant Clostridium perfringens β2 toxin protein;

[0048] Design primers according to the gene sequence of Clostridium perfringens β2 toxin reported in the literature

[0049] cpb2-PF: 5`CggAATTCTAATgAAAgAAATCgACgCTTAT3` and

[0050] cpb2-RF:5`GAATgCggCCgCTgCACAATACCTTCACC3`,

[0051] The cpb2 gene was amplified from the C-type standard strain of Clostridium perfringens, constructed into pTIG-Trx vector, transformed into BL21(DE3) strain, and labeled as BL21(DE3)pTIG-cpb2.

[0052] Take the BL21(DE3)(pTIG-cpb2) frozen strain and streak it on the LB solid medium containing ampicillin, cultivate overnight, pick a single colony and culture it in a shaker flask, culture it until the OD600 is 0.6-0.8, add the final The expression was induced by IPTG at a concentration of 1 mmol / L, and the expression was induced at 37°C for 3 hours, and the cells were harvested by centrifugation at 8000 rpm.

Embodiment 2

[0053] Embodiment 2: Purification of soluble recombinant Clostridium perfringens β2 toxin protein;

[0054] The purification method of the soluble Clostridium perfringens β2 toxin recombinant protein is as follows: centrifuge to collect 800mL recombinant expression cells after induction, wash once with PBS, add 1 / 10 of the original volume of the cell pellet to resuspend in PBS, and ultrasonically break for 10 minutes Afterwards, centrifuge at 8000r / min for 5 minutes, take the supernatant sample and use a filter with a pore size of 0.22 μm to remove the coarse suspended matter, which can be used as the loading sample of the AKTA protein purification system; -In the NTA gel column, after adding all the samples, add 500mM imidazole eluent to elute the His-tagged protein, harvest the peak protein eluted with 100mM imidazole, add it to the dialysis bag, and place it in PBS solution for dialysis at 4°C overnight.

Embodiment 3

[0055] Example 3: Mass spectrometric identification of soluble recombinant Clostridium perfringens β2 toxin protein;

[0056] The purified recombinant Clostridium perfringens β2 toxin protein was subjected to SDS-PAGE electrophoresis, and the target band at 28kDa on the PAGE gel was cut out for mass spectrometry analysis. The amino acid sequence after splicing was detected by peptide mass spectrometry. Protein BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) comparison results showed that the amino acid sequence of the target protein had the most similar matching degree with Clostridium perfringens β2 toxin, indicating that BL21(DE3 ) (pTIG-cpb2) expression and purification of the obtained target protein is recombinant Clostridium perfringens β2 toxin protein.

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Abstract

The invention discloses an indirect ELISA (enzyme-linked immuno sorbent assay) detection method for a clostridium perfringens beta 2 toxin antibody. The indirect ELISA detection method comprises the following steps: step 1, establishing an indirect ELISA method for the clostridium perfringens beta 2 toxin antibody; and step 2, determining a result judgment standard. The invention belongs to the technical field of pathogenic microorganism detection, and particularly provides an indirect ELISA system for detecting a clostridium perfringens beta2 toxin antibody, which is good in specificity, high in sensitivity and good in repeatability, and provides an effective detection means for the clostridium perfringens infection condition of animals. The indirect ELISA detection method for the clostridium perfringens beta2 toxin antibody has a wide application prospect in the aspects of clinical serum epidemiological investigation and vaccine immune effect evaluation, and can be used for rapidly detecting the clostridium perfringens beta2 toxin antibody.

Description

technical field [0001] The invention belongs to the technical field of pathogenic microorganism detection, and specifically refers to an indirect ELISA detection method for Clostridium perfringens β2 toxin antibody. Background technique [0002] Clostridium perfringens (Clostridium perfringens) causes human and animal food poisoning, enterotoxemia, necrotic enteritis and gas gangrene, which is a kind of worldwide zoonotic infectious disease, which has been subject to domestic the general attention of foreign researchers. [0003] The pathogenic factors of the bacteria are mainly 18 kinds of exotoxins and enzymes secreted by the bacteria; in 1997, Gibert of the Pasteur Institute in France found a lethal Clostridium perfringens isolated from a strain of piglet enteritis. Sexual exotoxin, named β2 toxin; the molecular weight of the toxin protein is 28kD, the isoelectric point is 5.4-5.5, and it is sensitive to protease. In 2015, Fisher et al. used the PCR detection method to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/577G01N33/56911
Inventor 曾瑾王玉炯吴霜宋孚洋李勇马臣杰张思雨马玲玲马红梅王东李文
Owner NINGXIA UNIVERSITY
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