Epitope polypeptide of dengue virus type 2 NS3 protein and application thereof
A technology of epitope polypeptide, dengue virus, applied in the field of medicine
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0025] Embodiment 1, the preparation of material
[0026] Dengue virus type 2 (DV2, Tr1751 strain): preserved by our laboratory.
[0027] Experimental animals: BALB / c mice, 6-8 weeks old, SPF grade, female, weighing 18-22 g, purchased from the Experimental Animal Center of Third Military Medical University.
[0028] Synthetic peptide: synthesized by Shanghai Sangon Bioengineering Co., Ltd., dissolved in dimethyl sulfoxide (DMSO) to a concentration of 5 mg / mL, stored at -70°C, and diluted to 1 mg / mL with phosphate buffered saline (PBS) before use.
[0029] Freund's adjuvant and Freund's incomplete adjuvant were purchased from Sigma, USA; phage peptide library display kit (Ph.D.-7 TM Phage Display Peptide Library Kit), purchased from NEB, USA.
[0030] LB liquid medium: tryptone 10g, yeast extract 5g, NaCl 10g, add distilled water to 1000mL, adjust pH to 7.4, and autoclave sterilization.
[0031] LB solid medium: add 1.5g agar powder to 100mL LB culture medium, and pour th...
Embodiment 2
[0053] Embodiment 2, antibody purification
[0054] (1) Preparation of buffer:
[0055] Equilibrium buffer: 50mM Tris-Cl, 3M NaCl, pH7.8-8.5;
[0056] Elution buffer: 0.1M sodium citrate buffer, pH4.0;
[0057] Regeneration buffer: 0.1M sodium citrate buffer, pH3.0;
[0058] Neutralization buffer: 1M Tris-Cl, pH9.0;
[0059] The above buffers need to be filtered with a 0.45 μm filter membrane before use.
[0060] (2) Sample preparation
[0061] Splenocytes from BALB / c mice immunized with dengue virus type 2 (Tr1751 strain) were fused with myeloma cell SP20, and the positive hybridoma cells prepared were injected into the peritoneal cavity of BALB / c mice to obtain ascites (see Zongtao Chen et al. al. Production of a Monoclonal Antibody Against Non-Structural Protein 3 of Dengue-2 Virus by Intrasplenic Injection). The obtained ascitic fluid was diluted 10 times with the equilibrium buffer to ensure that the composition and pH of the sample liquid were close to the equil...
Embodiment 3
[0069] Example 3. Using phage peptide library to screen antigen epitopes
[0070] 1. Phage screening
[0071] Day 1: Purified 4F5 antibody coated 96-well microplate
[0072] 1. Dissolve the 4F5 antibody prepared in Example 2 in 0.1M NaHCO of pH8.6 3 , prepare a 4F5 antibody solution with a concentration of 100 μg / mL.
[0073] 2. Add 150 μL of the 4F5 antibody solution prepared in step 1 to each well of the 96-well microplate, and rotate repeatedly until the surface is completely wet (be careful not to splash the solution).
[0074] 3. Then put the 96-well microplate into a humidified container (such as a sealable plastic box lined with wet paper towels), and incubate overnight at 4°C with slight shaking.
[0075] the next day
[0076] 4. Pick the single clone of host bacterium ER2738 (the plate that was spread during the phage titer determination) in 20mL LB liquid medium, and culture it with vigorous shaking at 37°C.
[0077] 5. Pour off the coating solution in the...
PUM
| Property | Measurement | Unit |
|---|---|---|
| pore size | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More