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Mycoplasma hyopneumoniae indirect ELISA antibody detection kit and application

A technology of mycoplasma hyopneumoniae and DNA sequence, which is applied in the detection field of animal virology and zoonosis, and achieves the effect of short detection time, broad market prospect and strong specificity

Active Publication Date: 2013-04-03
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The main purpose of the present invention is to overcome the deficiencies in the prior art and provide a Mycoplasma hyopneumoniae indirect ELISA antibody detection kit to solve the detection of Mycoplasma hyopneumoniae antibodies in pigs

Method used

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  • Mycoplasma hyopneumoniae indirect ELISA antibody detection kit and application
  • Mycoplasma hyopneumoniae indirect ELISA antibody detection kit and application
  • Mycoplasma hyopneumoniae indirect ELISA antibody detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Cloning of gene fragments of Mycoplasma hyopneumoniae p46 protein and p65 protein

[0068] 1. For the cloning method of the p46 and p65 protein gene fragments of Mycoplasma hyopneumoniae, see figure 2 shown.

[0069] The primers required to prepare the p46 gene fragment capable of prokaryotic expression are listed in Table 1.

[0070] Table 1 prepares the primers required for the p46 gene fragment capable of prokaryotic expression

[0071]

[0072] Note: The underlined part of the primers shown in Table 1 is the restriction site.

[0073] Table 2 prepares the primers required for the p65 gene fragment capable of prokaryotic expression

[0074]

[0075] Note: The underlined part of the primers shown in Table 2 is the restriction site.

[0076] 2. Preparation of p46 and p65 gene fragments capable of prokaryotic expression

[0077]Genomic DNA was extracted from the live vaccine of Mycoplasma hyopneumoniae (purchased from Nanjing Tianbang Biotechnology...

Embodiment 2

[0084] Example 2 Identification of recombinant strains pGEX-KG-46 and pGEX-KG-65 expressing p46 and p65 fusion proteins

[0085] Inoculate the strain with the correct mutation into the LB liquid medium containing 100mg / ml ampicillin at a ratio of 1:100 (volume ratio), shake and culture at 200r / min at 37°C for 3 hours, then inoculate again at a volume ratio of 1:1000 Add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.8mmol / L, and induce expression for 4 hours. Collect the bacteria and use 10mL Buffer A (recipe: weigh 6.057g Tris base, 0.1861g disodium edetate, 2.922g sodium chloride, 50mL glycerin, dissolve in deionized water and dilute to 1000mL, and use 0.22μm Store at 2-8°C after filtering through a filter membrane, and resuspend in 0.5mM DTT when used. After repeated freezing and thawing three times, perform ultrasonic crushing (UP 200S ultrasonic processor-manufactured by Dr. Hielscher, Germany, power: 200W, amplitude: 60%, operating frequency: 2...

Embodiment 3

[0086] Example 3 Preparation and testing of antigen

[0087] 1. Purification and inspection of p46 and p65 fusion proteins

[0088] GST-46 and GST-65 expressed in soluble form were purified by agarose-4B affinity layer suction column: add the supernatant to a centrifuge tube containing about 1 mL of GST-4B stock solution, place on a horizontal shaker at room temperature or After shaking slowly for 1 h under lower conditions, centrifuge at 2100 r / min for 5 min, transfer the supernatant to another centrifuge tube, add at least 10 times the volume of PBS with pH 7.4 (recipe: weigh 8.18 g sodium chloride, 0.20 g Potassium chloride, 1.42g disodium hydrogen phosphate, 0.245g potassium dihydrogen phosphate, dissolve in deionized water, adjust pH to 7.4 with concentrated hydrochloric acid, dilute to 1000mL with deionized water, then filter with 0.22μm filter membrane Store at room temperature.) Shake on a shaker for about 10 minutes, centrifuge at 2100r / min for 5 minutes, wash twice,...

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Abstract

The invention belongs to the technical field of animal virology and animal infectious diseases detection, and concretely relates to a Mycoplasma hyopneumoniae indirect ELISA antibody detection kit and an application. According to the invention, two strains of recombinant E. coli pGEX-KG-46 and pGEX-KG-65 expressing M. hyopneumoniae p46 protein and p65 protein are obtained through a gene engineering recombinant technology. The kit provided by the invention comprises an ELISA plate using expression proteins of mutant P46 gene and P65 gene of the Mycoplasma hyopneumoniae membrane proteins as common antigen coating, and other core reagents. The invention discloses clone of the P46 gene and the P65 gene of the Mycoplasma hyopneumoniae membrane proteins, site-directed mutagenesis, and expression and purification methods of the p46 protein and the p65 protein, and further discloses a Mycoplasma hyopneumoniae indirect ELISA antibody detection method. The indirect ELISA antibody detection kit provided by the invention can be used for a large-scale clinical testing and an epidemiological investigation of the Mycoplasma hyopneumoniae antibody, and has a wide market prospect.

Description

technical field [0001] The invention relates to the technical field of animal virology and animal infectious disease detection. Specifically, the present invention relates to an indirect ELISA antibody detection kit for Mycoplasma hyopneumoniae and its application in detecting Mycoplasma hyopneumoniae antibody in swine herds. Background technique [0002] Mycoplasma swine pneumonia, commonly known as swine panting disease or porcine endemic pneumonia, is widely distributed all over the world and is characterized by chronic, contact, highly contagious, high morbidity and low mortality, mainly manifested as cough and dyspnea. Visible lung tissue showed fleshy or marble-like lesions. [0003] Mycoplasma hyopneumoniae is one of the important pathogens of porcine respiratory disease syndrome, and can be secondary infected with PRRSV and PCV 2 etc., resulting in the failure of vaccination of various vaccines. Mycoplasma hyopneumoniae can be transmitted through the air. In addit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/531
Inventor 何启盖马丰英李燕库旭钢邹浩勇陈焕春郭爱珍徐高原
Owner HUAZHONG AGRI UNIV
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