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Recombinant MSG1 protein monoclonal antibody and blocked ELISA (Enzyme-linked Immuno Sorbent Assay) method for detecting haemophilus parasuis mycoplasma specific antibody

A monoclonal antibody, Mycoplasma haemophilus technology, applied in the field of serology, can solve the problem of lack of MSG1 protein monoclonal antibody, and achieve the effect of good repeatability, good specificity, and stable antibody secretion ability

Inactive Publication Date: 2013-01-23
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the preparation of MSG1 protein monoclonal antibody at home and abroad, and there is no report on the establishment of related blocking ELISA detection methods

Method used

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  • Recombinant MSG1 protein monoclonal antibody and blocked ELISA (Enzyme-linked Immuno Sorbent Assay) method for detecting haemophilus parasuis mycoplasma specific antibody
  • Recombinant MSG1 protein monoclonal antibody and blocked ELISA (Enzyme-linked Immuno Sorbent Assay) method for detecting haemophilus parasuis mycoplasma specific antibody
  • Recombinant MSG1 protein monoclonal antibody and blocked ELISA (Enzyme-linked Immuno Sorbent Assay) method for detecting haemophilus parasuis mycoplasma specific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of Mycoplasma suis recombinant MSG1 protein and detection protein

[0038] 1.1 Modification and artificial synthesis of MSG1 gene

[0039] According to the codon preference of E.coli and the obstacles encountered in the translation of tryptophan encoded by UGA, the entire MSG1 gene sequence was modified according to the degeneracy of codons, and at the same time at the 5' end and 3' end of the gene KpnI and EcoRI restriction sites were added at the 'end respectively, and the sequence is SEQ ID NO.2. The whole gene sequence was synthesized by Shanghai Jierui Bioengineering Co., Ltd.

[0040] 1.2 Cloning and identification of MSG1 gene

[0041] The pBAD / HisB vector (Invitrogen Company) was digested with KpnI and EcoRI and recovered from the gel, then the recovered vector was ligated with the synthesized MSG1 gene, the ligated product was transformed into DH5α competent cells, and the transformed product was coated with ampicillin On the resistance...

Embodiment 2

[0050] Example 2 Preparation of recombinant MSG1 protein monoclonal antibody against Mycoplasma suis

[0051] 2.1 Animal immunity

[0052] Take healthy BALB / c female mice aged 6-8 weeks (purchased from Nanjing Qingzilan Technology Co., Ltd.), and immunize them by subcutaneous multipoint injection. 100μg, three free 200μg. The antigen was emulsified with an equal volume of complete Freund's adjuvant for the first immunization, and the antigen was emulsified with an equal volume of incomplete Freund's adjuvant for the second and third immunizations. The interval between each immunization was 14 days, blood was collected from the tail vein 10 days after the third immunization, and the antibody titer was determined. Mice with a titer above 1:10000 were injected intraperitoneally with 200 μg of non-emulsified antigen 3 days before cell fusion for booster immunization.

[0053] 2.2 Indirect ELISA screening method

[0054] The antigen (recombinant MSG1 protein, prepared and purifi...

Embodiment 3

[0085] Example 3 Establishment of ELISA Detection Method for Mycoplasma Suis Enzyme-labeled Monoclonal Antibody Blocking

[0086] 3.1 Monoclonal antibody purification and horseradish peroxidase (HRP) labeling

[0087] The prepared ascites was sent to KingScript Bioengineering Company for purification and labeling of monoclonal antibody. The purity of the antibody reaches 97.6%. The results of SDS-PAGE analysis of ascitic fluid before and after purification are shown in Figure 5 .

[0088] 3.2 Selection of optimal reaction conditions for blocking ELISA

[0089] 3.2.1 Selection of optimal coating concentration of antigen and optimal dilution of serum

[0090] According to the checkerboard method, the purified recombinant MSG1 protein was diluted to the final concentrations of 2.0, 1.0, 0.5, and 0.25 μg / ml with carbonate buffer solution of pH 9.6, and coated overnight at 4°C. Then wash with 0.05mol / LPBS containing 0.05% Tween 20 (referred to as PBST, pH 7.2) for 5min×3 times...

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Abstract

The invention discloses a recombinant MSG1 protein monoclonal antibody and a blocked ELISA (Enzyme-linked Immuno Sorbent Assay) method for detecting a haemophilus parasuis mycoplasma specific antibody. A hybridoma cell line 1A7 of an excretion haemophilus parasuis mycoplasma recombinant MSG1 protein monoclonal antibody is preserved with preservation number of CGMCC NO. 4860 and the preservation date of May 13th, 2011. The monoclonal antibody of the haemophilus parasuis mycoplasma recombinant MSG1 protein is excreted by the hybridoma cell line 1A7 with preservation number of CGMCC NO. 4860. The monoclonal antibody as well as the haemophilus parasuis mycoplasma MSG1 recombinant protein and an etiology protein are provided with good reaction specificities. According to the invention, the monoclonal antibody is applied to the blocked ELISA (Enzyme-linked Immuno Sorbent Assay) method for detecting the haemophilus parasuis mycoplasma specific antibody; and the detection condition is optimized through a large quantity of experiments so that the method has the advantages of good repeatability, sensitivity and specificity.

Description

technical field [0001] The invention belongs to the field of serology, and relates to a recombinant MSG1 protein monoclonal antibody and a blocking ELISA method for detecting the specific antibody of swine haemophilus mycoplasma. Background technique [0002] The currently reported methods for detecting serum antibodies to M. suis mainly include complement fixation test, indirect hemagglutination test and enzyme-linked immunosorbent assay (ELISA). Among them, the ELISA method has the characteristics of high throughput and easy standardization, and has become a routine diagnostic reagent. first choice. Among the ELISA methods for detecting pathogenic antibodies, the monoclonal antibody blocking ELISA has good specificity because it can eliminate the interference of foreign protein components in the coated antigen. Nowadays, many commercial kits such as swine fever diagnostic kit (IDEXX), pseudorabies Diagnostic kits, etc. (IDEXX) all use the blocking ELISA method and are wid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20
Inventor 李玉峰张长莹姜平
Owner NANJING AGRICULTURAL UNIVERSITY
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