Adhesion protein Apd and application thereof in preparation of haemophilus parasuis indirect ELISA antibody detection kit

An adhesin and protein technology, applied in the fields of biotechnology and zoonotic diseases, which can solve problems such as false positives

Active Publication Date: 2018-12-18
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, such antibody detection methods or kits will inevitably cross-react with pathogens with similar antigenic components, resulting in false positives

Method used

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  • Adhesion protein Apd and application thereof in preparation of haemophilus parasuis indirect ELISA antibody detection kit
  • Adhesion protein Apd and application thereof in preparation of haemophilus parasuis indirect ELISA antibody detection kit
  • Adhesion protein Apd and application thereof in preparation of haemophilus parasuis indirect ELISA antibody detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Synthesis or cloning of embodiment 1 adhesin gene apd

[0051] (1) Synthesis of adhesin gene apd

[0052] According to the nucleotide sequence shown in SEQ ID No.1, the gene apd is synthesized.

[0053] (2) Cloning of adhesin gene apd

[0054] Primer pairs were designed based on the genome of Haemophilus parasuis isolate CF7066 (preserved by teacher Xu Xiaojuan of Huazhong Agricultural University):

[0055] P1,5'-CG GAATTC (EcoRI)GGAAACTTATATTGTTACTGGTGAA-3',

[0056] P2, 5'-CC CTCGAG (Xho I)GGTGATTGTGATTTTATTGTGGT-3';

[0057] The genome of Haemophilus parasuis isolate CF7066 was used as a template for PCR amplification. The PCR system was as follows: 31.5 μL deionized water, 5 μL 10×Pyrobest Buffer II, 4 μL dNTP Mixture (2.5 mM), 2 μL P1 (10 μM / L), 2 μL P2 (10 μM / L), 0.5 μL Pyrobest DNA Polymerase (Takara), 5 μL Haemophilus parasuis genomic DNA (150 ng / μL); PCR reaction program: pre-denaturation at 94°C for 5 min, enter cycle, denaturation at 94°C for 30 second...

Embodiment 2

[0058] The preparation of embodiment 2 adhesin protein

[0059] (1) Acquisition of adhesin gene

[0060] The present invention obtains a DNA fragment of 1577 nucleotides by DNA synthesis; or through the PCR primers, system and reaction procedures described in Example 1, amplifies the DNA fragment of 1577bp from Haemophilus parasuis isolate CF7066. The DNA fragment is named apd, and its nucleotide sequence is shown in SEQ ID No.1.

[0061] (2) Construction of adhesin expression plasmid

[0062] Dissolve the synthesized DNA fragments with deionized water; or recover PCR amplified fragments. The DNA fragment and vector pET-25b (Novagen) were double digested with EcoR I and Xho I, and the digested product was recovered and ligated with the vector, and the ligated product was transformed into Escherichia coli DH5α, and cultured overnight at 37°C until a single colony grew. Pick a single clone to extract the plasmid, and store the correctly sequenced pET-Apd plasmid at -20°C for ...

Embodiment 3

[0079] Example 3 Evaluation of recombinant adhesin protein as coating antigen of Haemophilus parasuis indirect ELISA antibody detection kit

[0080] (1) Expression of three recombinant proteins from Haemophilus parasuis

[0081] The extracellular regions of three outer membrane proteins of Haemophilus parasuis were selected and their prokaryotic expression plasmids pET-Ag2, pET-Ag3 and pET-Apd were constructed. Three plasmids were used to transform Escherichia coli BL21 (DE3), and induced for different times at different temperatures and different concentrations of IPTG to determine the optimal conditions for soluble expression of the target protein. The results showed that the optimal expression condition of rAg2 (recombinant Ag2) protein was 16°C, induced by 0.2mM IPTG for 12h; rAg3 (recombinant Ag3) protein was 16°C, induced by 0.2mM IPTG for 12h; The optimal expression condition is 37°C, 0.8mM IPTG induction for 4h ( figure 1 ).

[0082](2) Purification of three recomb...

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Abstract

The invention discloses an adhesion protein Apd and application thereof in preparation of a haemophilus parasuis indirect ELISA antibody detection kit. The invention verifies that the adhesion proteinApd is located on an outer membrane of the haemophilus parasuis, is expressed in 15 known serotypes respectively but does not exist in other bacterial pathogens of a pig, so that the adhesion proteinApd has high sensitivity and specificity when being taken as a diagnostic antigen. By optimizing expression and purification methods of a recombinant adhesion protein, a recombinant protein with goodimmunogenicity and reactionogenicity is obtained. By optimizing components and dosages as well as reaction conditions and result determination criterions of an indirect ELISA method taking the recombinant adhesion protein as an envelope antigen, the haemophilus parasuis indirect ELISA antibody detection kit is prepared. Animal experiments verify that the kit can be used for antibody detection after vaccine immunity and bacterial infection of the haemophilus parasuis. In comprehensive control and seroepidemiological investigation of a haemophilus parasuis disease, the kit can have a broad application prospect.

Description

technical field [0001] The invention relates to the fields of biotechnology and animal infectious diseases, in particular to an adhesin protein Apd and its application in the preparation of an indirect ELISA antibody detection kit for Haemophilus parasuis. Background technique [0002] Haemophilus parasuis (HPS) is a common bacterium in the upper respiratory tract of pigs, belonging to the genus Haemophilus in the Pasteurellaceae family, and is a small spherical, club-shaped to filamentous Gram-negative bacterium. HPS can cause Haemophilus parasuis disease (also known as Glasser's disease) in pigs when they are immunocompromised or stressed. Acute infection of HPS can lead to polyserositis, arthritis and meningitis, sepsis and death; chronic infection can cause suppurative rhinotracheitis and pneumonia. HPS is often co-infected with Escherichia coli, Streptococcus suis, and Pasteurella multocida, complicating the disease. The disease occurs and is popular in pig-raising co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569
Inventor 徐晓娟蔡旭旺刘云宝齐毅杜钰娇张勤学王湘如何启盖陈焕春
Owner HUAZHONG AGRI UNIV
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