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Anti-swine SC protein monoclonal antibody and application of monoclonal antibody in preparing mycoplasma hyopneumoniae SIgA antibody ELISA detection kit

A technology of Mycoplasma hyopneumoniae and monoclonal antibody, which is applied in immunoglobulin, biochemical equipment and methods, measuring devices, etc., can solve problems such as inapplicability for early diagnosis, and achieve high sensitivity, reduction of stress, and high stability. sexual effect

Active Publication Date: 2015-09-02
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the ELISA of existing serum antibodies is not suitable for the early diagnosis of the disease and the monitoring of immune indicators after active immunization

Method used

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  • Anti-swine SC protein monoclonal antibody and application of monoclonal antibody in preparing mycoplasma hyopneumoniae SIgA antibody ELISA detection kit
  • Anti-swine SC protein monoclonal antibody and application of monoclonal antibody in preparing mycoplasma hyopneumoniae SIgA antibody ELISA detection kit
  • Anti-swine SC protein monoclonal antibody and application of monoclonal antibody in preparing mycoplasma hyopneumoniae SIgA antibody ELISA detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 prepares porcine recombinant SC protein

[0033] 1. RNA Extraction of Porcine SC Protein

[0034] The samples were porcine intestinal epithelial cells, tracheal epithelial cells or lymphocytes, or a mixture of the three.

[0035] Porcine small intestinal mucosal epithelium (tracheal epithelium) treatment: carefully scrape the porcine small intestinal mucosal epithelium (tracheal epithelium), cut into pieces, add 500 μl of autoclaved PBS buffer (0.01M, pH7.2-7.4) and homogenate ;

[0036] Treatment of lymphocytes: take 500 μl of pig blood, add 500 μl of lymphocyte separation medium (purchased from Shanghai Huajing Bio-Technology Co., Ltd.), centrifuge (20 min at 2000 rpm), and precipitate with 500 μl of PBS buffer (0.01 M, pH7.2-7.4) resuspended;

[0037] The processed samples were extracted by conventional methods for total cellular RNA

[0038] 2. Amplification of porcine SC protein gene fragment

[0039] (1) RT-PCR (all reagents used were purchased f...

Embodiment 2

[0082] Example 2 Preparation of anti-pig SC protein monoclonal antibody

[0083] 1. Mice Immunization

[0084] The porcine recombinant SC protein purified in Example 1 was mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, USA), and fully emulsified, and subcutaneously immunized five 8-week-old BALB / c mice (Comparative Medicine of Yangzhou University) center), the dose of antigen inoculation was 100 μg / rat. Two weeks after the first immunization, the same dose of porcine recombinant SC protein was emulsified with Freund's incomplete adjuvant (Sigma-Aldrich, USA) to immunize the mice in the same way, and then the mice were immunized in the same way every 2 weeks, and repeated 3 times until the serum The titer reaches above 1:6400. 3-5 days before cell fusion, intraperitoneal injection of double the amount of antigen without adjuvant booster immunization once, cell fusion can be carried out.

[0085] 2. Preparation of sp 2 / 0 myeloma cells

[0086] The s...

Embodiment 3

[0123] Example 3 Mycoplasma hyopneumoniae SIgA antibody ELISA detection kit

[0124] Mycoplasma hyopneumoniae SIgA antibody ELISA detection kit contains:

[0125] 1. Preparation of antibody detection plate coated with Mycoplasma hyopneumoniae P97R1 protein by antibody capture method

[0126] (1) Preparation of Mycoplasma hyopneumoniae P97R1 protein:

[0127] According to the literature (Liu Maojun, Shao Guoqing, Zhang Ying, Nie Xiangting. Cloning and expression of the R1 region of the epitope of the Mycoplasma hyopneumoniae P97 gene. Jiangsu Agricultural Journal. 2005.21(3):207-11) to construct the antigenic determinant of the P97 gene carrying the Mycoplasma hyopneumoniae Recombinant strains of gene fragments in the R1 region. The recombinant bacteria were inoculated into LB liquid medium containing ampicillin, and cultivated in a shaker at 37°C and 180rpm until the OD 600 = 0.6 to 0.8, add IPTG with a final concentration of 0.1mmol / L to induce expression, continue to cult...

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Abstract

The invention provides an anti-swine SC protein monoclonal antibody and an application of the monoclonal antibody in preparing a mycoplasma hyopneumoniae SIgA antibody ELISA detection kit, and relates to the technical field of animal virological and epizootiological detection. The anti-swine SC protein monoclonal antibody is secreted by a hybridoma cell strain 4H11 and the collection number of the anti-swine SC protein monoclonal antibody is CCTCC NO: C201526. The invention also discloses the monoclonal antibody and the application of the monoclonal antibody in preparing the mycoplasma hyopneumoniae SIgA antibody ELISA detection kit. The kit has high specificity, high stability and high sensitivity; a detection sample can be sampled conveniently; and the kit is capable of distinguishing porcine mycoplasma pneumonia inactivated vaccine immunization and natural infection, and can be applied to the early diagnosis of pneumonic porcine mycoplasma infection and the evaluation of the immune effect after attenuated live vaccine immunization.

Description

technical field [0001] The invention relates to the technical field of animal virology and animal infectious disease detection, in particular to an anti-pig SC protein monoclonal antibody and its application. Background technique [0002] Mycoplasma hyopneumoniae (Mhp) is the pathogen that causes mycoplasma pneumonia (MPS), commonly known as swine asthma. Porcine asthma is one of the most widespread, fastest-spreading, and most difficult to purify diseases in pig herds. Mycoplasma hyopneumoniae spreads through the respiratory tract. After infecting the respiratory epithelium, the cilia of the respiratory tract will shrink, fall off, and be damaged. [0003] Studies have shown that the prevention of MPS is mainly through local cellular immunity and mucosal immunity in the respiratory tract. SIgA (secretory immunoglobulin A) is a major effector involved in mucosal immune responses. They are important components of the body's immune system and play an important role in the i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42C12N5/20G01N33/577
Inventor 冯志新白昀邵国青王海燕刘茂军熊祺琰马庆红甘源
Owner JIANGSU ACAD OF AGRI SCI
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