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Haemophilus parasuis (Hps) immunoprotecive antigen OppA

A technology of Haemophilus suis and protective antigen, applied in the field of animal infectious disease subunit vaccine preparation

Active Publication Date: 2013-09-25
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, oligopeptide-binding protein (OppA) can bind 2-5 disabled peptides without selectivity

Method used

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  • Haemophilus parasuis (Hps) immunoprotecive antigen OppA
  • Haemophilus parasuis (Hps) immunoprotecive antigen OppA
  • Haemophilus parasuis (Hps) immunoprotecive antigen OppA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the discovery of OppA protein immunogenicity

[0034] 1. Preparation of secreted protein samples

[0035] The strain of Haemophilus parasuis (Hps) (purchased from the National Center for Veterinary Microorganism Culture Collection, strain number: CVCC3361) was taken out from the refrigerator at -70°C, thawed at room temperature, and then streaked and inoculated in TSA (Becton, Dickinson and Company) solid medium (containing 10% horse serum, 0.01% NAD (nicotinamide adenine dinucleotide)), cultivated at 37°C for 36h, then picked a single colony and inoculated it in TSB (Becton, Dickinson and Company) liquid Culture medium (without horse serum, containing 0.02% NAD), cultured at 37°C for 24 hours, and then transferred to 500 mL TSB liquid medium (without horse serum, containing 0.02% NAD) with a volume of 1% bacterial solution, 37 Cultivate for about 16 hours at ℃ to make the OD of the bacterial solution reach 1.0. Add protease inhibitors to the bacterial c...

Embodiment 2

[0049] Embodiment 2, the expression of OppA protein

[0050] 1. Extraction of total Hps DNA

[0051] Centrifuge 1 mL of the overnight culture of Hps (CVCC3361) at 12,000 rpm for 1 minute, and discard the supernatant. Add 40 μL DB solution (TIANamp Bacteria DNA Kit, Tiangen Biological Co., Ltd.), 160 μL lysozyme and 8 μL RNaseA to the cell pellet. Shake vigorously to mix well. Incubate at 37°C for 30-60 minutes, and invert the centrifuge tube several times. Add 200 μL of DLT solution (TIANamp Bacteria DNA Kit, Tiangen Biological Co., Ltd.) and 25 μL of proteinase K (TIANamp Bacteria DNA Kit, Tiangen Biological Co., Ltd.), and immediately mix it gently by inversion. Place in a 65°C water bath for at least 30 minutes, and invert the centrifuge tube several times. Centrifuge at 12000rpm for 3-5 minutes, and pipette all the supernatant into a clean centrifuge tube. Add 200 μL of absolute ethanol, mix well, suck it into the adsorption column, centrifuge at 12000 rpm for 30 seco...

Embodiment 3

[0077] Example 3, Identification of OppA Protein Immunoprotection

[0078] 1. Effect of Hps on LD of Kunming mice 50 Determination of

[0079] Inoculate Hps (purchased from the National Center for Veterinary Microorganism Culture Collection, strain number: CVCC3361) in TSB liquid medium (containing 10% horse serum, 0.01% NAD) at 37°C at 200 rpm / min for 14 to 16 hours, and then the next day Spread TSA solid medium (containing 10% horse serum, 0.01% NAD) and incubate at 37°C for 24-36h. Wash the bacterial lawn with PBS and dilute to 5×10 8 CFU / mL (OD 600 about 1.0), then 2-fold concentrated step by step, concentrated to the required dose (as shown in Table 1) and then injected into mice. Female Kunming mice aged 8-10 weeks (purchased from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were divided into 5 groups, 10 mice in each group. Each mouse was intraperitoneally injected with 100 μL of the challenge dru...

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Abstract

The invention relates to identification of a Haemophilus parasuis (Hps) immunoprotecive antigen, separation and cloning of a protein gene and application of a protein coded by the Hps immunoprotecive antigen in a vaccine. According to the invention, a new protein OppA having immunogenicity is separated from Hps (the culture collection number is CVCC3361), and the nucleotide sequence is shown as SEQ ID NO:2 in a sequence table and is formed by coding 545 amino acids. The OppA is a new protein having immunogenicity and can provide effective immunoprotection for Hps infection in mice. The invention also comprises preparation of an Escherichia coli recombinant bacterium BL21 / Hps-oppA expressing the immunogenicity protein gene oppA. The recombinant Hps OppA protein expressed by the invention has favorable safety and protection efficacy, and the immunoprotection effect is up to 60%.

Description

technical field [0001] The invention relates to the technical field of preparation of animal infectious disease subunit vaccines. It specifically relates to the identification of an immunoprotective antigen of Haemophilus parasuis, the isolation and cloning of the protein gene and the application of the encoded protein in vaccines. Background technique [0002] The combination of modern proteomics technology and traditional immunohybridization method produces a new interdisciplinary subject - Immunopreteomics (Immunoproteomics), which is rapidly applied to the research of immunogenic proteins of humans, animals, plants and microorganisms. A powerful tool for large-scale screening of pathogen antigens. In this study, the secretome of Haemophilus parasuis (Hps) was analyzed by two-dimensional electrophoresis and Western blot technology, and antigenic proteins with good antigenicity and strong protection were screened out, in order to develop serological diagnostic markers and...

Claims

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Application Information

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IPC IPC(8): C07K14/285C12N15/31C12N15/63C12N1/21A61K39/102A61K48/00A61P31/04
Inventor 王春来李刚张艳禾谢芳
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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