Haemophilus parasuis (Hps) immune protective antigen CdtA
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.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0032] Embodiment 1, the cloning of CdtA 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 CdtA protein
[0054] 1. Preparation and purification of CdtA protein
[0055] The engineered bacteria prepared in Example 1 (four) were cultured in LB medium containing 50 μg / ml kanamycin, cultivated at 37° C. for 3 h; OD 600 When = 0.7, add IPTG to a final concentration of 0.8 μM, transfer to 37° C. and continue culturing for 4 h.
[0056] Collect the bacteria by centrifugation at 5000rpm for 10 minutes, suspend in PBS solution, 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 wit...
Embodiment 3
[0061] Example 3, Identification of CdtA Protein Immunoprotection
[0062] 1. Effect of Hps on LD of Kunming mice 50 Determination of
[0063] Inoculate Hps (purchased from the National Veterinary Microorganism Culture Collection Center, strain number: CVCC3361) in TSB liquid medium (containing 10% horse serum, 0.01% NAD) at 37°C at 200rpm / min for 14-16 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 drug, obser...
PUM
| Property | Measurement | Unit |
|---|---|---|
| Molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More