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Haemophilus parasuis (Hps) immunoprotecive antigen CdtB

A technology of Haemophilus suis and protective antigen, which is applied in the field of animal infectious disease subunit vaccine preparation, can solve the problems that it cannot cause the expansion and apoptosis of target cells, and the whereabouts are not clear.

Inactive 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

The DNase activity of the CdtB subunit is the key to the cytotoxic effect of CDT holotoxin. CdtA and CdtC have no DNase-like activity and cannot cause the expansion and apoptosis of target cells. The main function of the two is to bind to the surface of the cell membrane and promote CdtB. Enter the nucleus to exert their cytotoxic effects, but how they do so and where they go after their mission remains unclear

Method used

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

Examples

Experimental program
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Embodiment 1

[0032] Embodiment 1, the cloning of CdtB protein coding gene

[0033] 1. Extraction of total Hps DNA

[0034] Centrifuge 1 mL of the overnight culture solution of Haemophilus parasuis (Hps) (purchased from the National Center for Veterinary Microorganisms, strain number: 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 centrifug...

Embodiment 2

[0053] Embodiment 2, expression and purification of CdtB protein

[0054] 1. Preparation and purification of CdtB protein

[0055] Cultivate the engineered bacteria prepared in Example 1 (IV) in LB medium containing 50 μg / ml kanamycin, and culture at 37°C for 3h; when OD600=0.7, add IPTG to a final concentration of 0.8μM, and transfer to 37°C Continue to cultivate for 4h.

[0056] Collect the bacteria by centrifugation at 5000rpm for 10 minutes, suspend in the solution in PBS, and ultrasonically break in an ice bath (300w, 10 minutes; ultrasonic for 3s, stop for 5s), then centrifuge at 15000rpm for 10 minutes to collect the precipitate, and dissolve the precipitate with 30ml of solution A ( NaH 2 PO 4 100mmol / L, Tris-HCl10mmol / L, 8mol / L urea to adjust the pH to 8.0) to dissolve, and can be briefly ultrasonicated to induce dissolution. Add 2 mL of pre-treated resin (Ni-NTA His-Bind Resin, Novagen), shake at a low speed at 4°C for 30 minutes to fully combine the resin with t...

Embodiment 3

[0061] Example 3, Identification of CdtB Protein Immunoprotection

[0062] 1. Effect of Hps on LD of Kunming mice 50 Determination of

[0063] 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 CdtB 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 277 amino acids. The CdtB 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-CdtB expressing the immunogenicity protein gene CdtB. The recombinant Hps CdtB protein expressed by the invention has favorable safety and protection efficacy, and the immunoprotection effect is up to 70%.

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] Cell lethal expansion toxin is a newly discovered member of the bacterial protein toxin family. It was first discovered in the culture supernatant of Escherichia coli. It acts on Chinese hamster ovary cells (Chinese hamster ovary cells, CHO) in vitro and can cause target cells to expand and die. Therefore, the toxin was named "cyto-lethal expansion toxin". Currently, CDT is the only toxin with DNase I activity among bacterial toxins, which has genotoxic effects, causes DNA double-strand breaks, and causes irreversible cell cycle arrest and apoptosis in mammalian cells. [0003] It has been confirmed that a variety o...

Claims

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Application Information

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IPC IPC(8): C07K14/285C12N15/31C12N15/70C12N1/21A61K39/102A61K48/00A61P31/04C12R1/21
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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