Antigen and indirect ELISA test kit for identification of Mycoplasma bovis vaccine strain or field strain infection

A detection kit, a technology for Mycoplasma bovis, which is used in biological tests, measuring devices, immunoassays, etc.

Active Publication Date: 2022-06-14
天康制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no accurate method for the differential diagnosis between its use and wild poison

Method used

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  • Antigen and indirect ELISA test kit for identification of Mycoplasma bovis vaccine strain or field strain infection
  • Antigen and indirect ELISA test kit for identification of Mycoplasma bovis vaccine strain or field strain infection
  • Antigen and indirect ELISA test kit for identification of Mycoplasma bovis vaccine strain or field strain infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Membrane protein (P275) gene, leucyl aminopeptidase (pepA) gene fragment PCR amplification

[0062] 1. Cultivation of Mycoplasma bovis strains: Inoculate the Mycoplasma strains isolated from diseased cattle by our company into PPLO liquid medium at 37°C and 5% CO 2 Cultivated in an incubator;

[0063] 2. Genomic DNA extraction: extract the genome according to the method provided by the Mycoplasma Genomic DNA Extraction Kit;

[0064] 3. Target gene amplification: Amplification system is 50 μL system: Taq enzyme (1000u / mL) 2 μL, 10×BUFFER 5 μL, dNTP (10 mM) 2 μL, upstream primer (10 μM) 2 μL, downstream primer (10 μM) 2 μL, template 10 μL , ddH 2 O 27 μL. PCR reaction conditions: pre-denaturation at 95°C for 5 minutes; 35 cycles of denaturation at 95°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 1 minute; extension at 72°C for 15 minutes; storage at 4°C.

[0065] 4. Purification of the target gene: PCR product gel electrophore...

Embodiment 2

[0066] Embodiment 2 connection and conversion

[0067] 1. Plasmid pET-30a (+), membrane protein (P275) gene, and leucyl aminopeptidase (pepA) gene were subjected to KpnI and BamHI double enzyme digestion;

[0068] 1.1 Perform 1% agarose gel electrophoresis on the digested product, recover the target band from the gel and purify it;

[0069] 1.2 According to the method steps provided by the T4 connection kit, connect the plasmid with the p275 and pepA genes;

[0070] 1.3 Kanamycin (100ml / L) solid medium screening P275-pET-30a (+), pepA-pET-30a (+) transformed into Escherichia coli BL21 positive strain;

[0071] 1.4 Identification of P275-pET-30a(+), pepA-pET-30a(+) recombinant prokaryotic expression vectors by double enzyme digestion.

[0072] 2. Prokaryotic expression and purification of P275-pET-30a(+), pepA-pET-30a(+) proteins

[0073] 2.1 Pick a single colony of Escherichia coli BL21 containing P275-pET-30a (+), pepA-pET-30a (+) expression vector in LB solid medium (cont...

Embodiment 3E

[0080] Embodiment 3ELISA detects bovine serum

[0081] Randomly select 20 cattle from each of the mycoplasma-negative cattle farm, the inactivated vaccine immunization experiment cattle farm, and the mycoplasma-positive cattle farm. Follow the indirect ELISA method to detect the OD of P275 protein and pepA protein antibody in serum 450nm values ​​(see Table 1 for the results).

[0082] 1. Take 5ml of blood from the neck of the cow, centrifuge at 1000g for 20min at 37°C for 2h, and store the supernatant at 4°C;

[0083] 2. Coating: Dilute P275 expression protein concentration to 50 μg / ml and pepA protein concentration to 80 μg / ml with coating diluent (PBS pH7.0), respectively add to different microwells of the ELISA plate, 100 μl per well, 4 ℃ 24h;

[0084] 3. Blocking: Discard the coating solution, add 250 μl of 5% protamine to each well, and keep at 37°C for 1 hour;

[0085] 4. Plate washing: Discard the blocking solution, add washing solution to fill the wells, vibrate f...

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Abstract

The invention relates to the field of vaccines, in particular, it provides an antigen for identifying the Mycoplasma bovis vaccine strain or wild strain infection and an indirect ELISA detection kit. The invention provides a group of antigens for identifying the Mycoplasma bovis vaccine strain or wild strain infection, including membrane protein fragments and leucyl aminopeptidase fragments. The inventor found through research that after cattle were infected with different pathogens (vaccine strains and wild strains), only the changes of membrane protein antibodies and leucyl aminopeptidase antibodies presented a certain regularity. Acylaminopeptidase antigen detects the content of bovine inner membrane protein antibody and leucylaminopeptidase antibody, so that the differential diagnosis of Mycoplasma bovis vaccine strain or wild strain infection can be realized, and it provides a basis for the identification of vaccine strains and wild strains after use. new ideas and techniques.

Description

technical field [0001] The invention relates to the field of vaccines, in particular to an antigen and an indirect ELISA detection kit for identifying the Mycoplasma bovis vaccine strain or wild strain infection. Background technique [0002] Mycoplasma bovis is an important pathogenic mycoplasma, which can cause bovine mastitis, bovine pneumonia, arthritis, reproductive tract inflammation, infertility and other diseases. After being infected with mycoplasma, cattle will be infected with other pathogens, which will affect the health of cattle. Production capacity seriously endangers the healthy development of animal husbandry, and has become one of the important pathogens that endanger the cattle industry. [0003] Mycoplasma bovis pneumonia caused by Mycoplasma bovis is a bovine respiratory infectious disease mainly characterized by necrotizing pneumonia. The body temperature of the sick cow rises to 42°C, the spirit is depressed, the appetite is reduced, panting, severe c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/30C12N9/48G01N33/68G01N33/569G01N33/58G01N33/543
CPCC07K14/30C12N9/485G01N33/6854G01N33/56933G01N33/581G01N33/54393C12Y304/11001G01N2469/20
Inventor 贺笋任立松郝成武潘毅平李延涛任郭子君
Owner 天康制药股份有限公司
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