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Haemophilus parasuis (Hps) subunit vaccine composition and application

A technology of Haemophilus suis and composition, which is applied in the field of subunit vaccine preparation of animal infectious diseases, and can solve the problems of inefficiency, uneconomical, time-consuming, etc.

Active Publication Date: 2014-01-01
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are many unfavorable reasons such as inefficiency, time-consuming and uneconomical in traditional research methods, which limit the discovery of high-efficiency vaccine targets to a certain extent.

Method used

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  • Haemophilus parasuis (Hps) subunit vaccine composition and application
  • Haemophilus parasuis (Hps) subunit vaccine composition and application
  • Haemophilus parasuis (Hps) subunit vaccine composition and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the discovery of protein immunogenicity

[0020] 1. Preparation of secreted protein samples

[0021] The Hps strain was taken out from the -70°C refrigerator, thawed at room temperature, and then inoculated in TSA (Becton, Dickinson and Company) solid medium (containing 10% horse serum, 0.01% NAD (nicotinamide adenine dinucleotide) ), cultured at 37°C for 36h, then picked a single colony and inoculated it in TSB (Becton, Dickinson and Company) liquid medium (without horse serum, containing 0.02% NAD), cultured at 37°C for 24h, and then inoculated with 1% bacterial solution The volume was transferred to 500mL TSB liquid medium (without horse serum, containing 0.02% NAD), and cultured at 37°C for about 16 hours, so that the OD of the bacterial solution reached 1.0. Add protease inhibitors to the bacterial culture and centrifuge at 5000g for 1h at 4°C. Take the supernatant and filter out the residual Hps with a 0.22 μm filter membrane. Add 1 / 6 volume of pr...

Embodiment 2

[0035] Embodiment 2, expression of seven kinds of proteins

[0036] 1. Extraction of total Hps DNA

[0037] Centrifuge 1 mL of the overnight culture of Hps (CVCC 3361) 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...

Embodiment 3

[0068] Embodiment 3, the identification of seven kinds of protein mixture immune protection

[0069] 1. Effect of Hps on LD of Kunming mice 50 Determination of

[0070] Haemophilus parasuis (Hps) (purchased from the National Center for Veterinary Microorganism Culture Collection, strain number: CVCC 3361) was inoculated in TSB liquid medium (containing 10% horse serum, 0.01% NAD) at 37°C Cultivate at 200 rpm / min for 14-16 hours, then apply TSA solid medium (containing 10% horse serum, 0.01% NAD) the next day and incubate at 37°C for 24-36 hours. 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 w...

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Abstract

The invention provides a haemophilus parasuis (Hps) immunoprotective antigenic composition. The composition comprises seven haemophilus parasuis immunoprotective antigens, wherein the amino acid sequences of the seven haemophilus parasuis immunoprotective antigens are shown as SEQ ID NO:1-7 respectively. A plurality of new proteins with immunogenicity, including HbpA, afuA, oppA, oppA2, D15, Hps06257 and nqrA, are separated from the haemophilus parasuis (strain collection number: CVCC 3361), the nucleotide sequences of the proteins are shown as SEQ ID NO:8-14 in a sequence table, and 531, 346, 545, 513, 417, 263 and 448 amino acids are coded respectively. The coded products of the genes are new proteins with immunogenicity, and the mixture thereof can provide effective immunological protection for mice infected with haemophilus parasuis. The mixture of the immunogenic proteins of the recombinant haemophilus parasuis expressed by the composition has good safety and protection effect, and the immunological protection effect of the composition reaches 80 percent.

Description

technical field [0001] The invention relates to the technical field of preparation of animal infectious disease subunit vaccines. In particular, it relates to an immunoprotective antigen composition of Haemophilus parasuis and its preparation method and application. The composition contains seven kinds of immune protective antigens of Haemophilus parasuis. Background technique [0002] Haemophilus parasuis (Hps) is a Gram-negative bacterium that often colonizes the upper respiratory tract of pigs. It is an opportunistic pathogen that can cause Haemophilus parasuis disease in pigs. The bacterium was first reported in 1910, so the disease is also known as Glasser's disease ( disease). Hps only infects pigs and can affect pigs aged 2 weeks to 4 months, and the onset is common in pigs aged 5 to 8 weeks. The incidence rate is 10% to 15%, and the mortality rate can reach 50% in severe cases, which brings huge losses to the pig industry. The main features of the disease are fi...

Claims

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Application Information

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IPC IPC(8): A61K39/116A61P31/04A61K39/102
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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