Nano toxoid vaccine and application thereof
A technology for toxoids and vaccines, applied in the directions of antitoxins, medical preparations with non-active ingredients, and medical preparations containing active ingredients, etc., can solve the problems such as the need to improve the ability of immune activation, the complex process, and the difficulty in large-scale production. The effect of preventing toxin damage and improving safety
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Embodiment 1
[0045] Embodiment 1: the structure of nano toxoid vaccine
[0046] In this embodiment, red blood cells are firstly extracted from whole blood, and the red blood cell membrane is separated and purified by hypotonic method and centrifugation. Specific operation: remove the eyeballs of six-week-old male ICR mice to take blood, anticoagulate the whole blood with heparin sodium, centrifuge at 700g for 10 minutes at 4°C, discard the upper plasma and buffy coat (white blood cells and platelets), and then use The bottom erythrocytes were resuspended in 1 mM EDTA in PBS and washed three times. Collect the red blood cells at the bottom and add an equal volume of PBS solution containing 1mM EDTA to make a red blood cell suspension, add each 250μL to a 1.5mL EP tube, then add 950μL of 0.2mM EDTA aqueous solution, mix well, vortex to break the red blood cells, and then add 50μL of 20 xPBS to adjust to isotonicity. Centrifuge at 20,000 g for 10 minutes at 4°C, discard the supernatant. Re...
Embodiment 2
[0055] Embodiment 2: Physicochemical property characterization of nano toxoid vaccine
[0056] The non-toxin-loaded erythrocyte membrane fusogenic liposomes (RM-PLs(-)) were used as the control. The average particle size, particle size distribution and polydispersity coefficient of RM-PLs (Hlα) were measured by dynamic light scattering method, and their Zeta potential was measured. The results showed that the average particle size of RM-PLs(Hlα) was 120nm, and the particle size distribution was narrow, and the potential was -36.5mV, which was similar to RM-PLs(-). After negative staining with uranium acetate, the morphology and size of RM-PLs (Hlα) were observed by transmission electron microscopy, and the results showed ( figure 2 B-D), RM-PLs (Hlα) are regular spherical and uniform in size, similar to RM-PLs (-). The above results indicated that the prescription amount of toxin loading had no significant effect on the physicochemical properties of erythrocyte membrane fus...
Embodiment 3
[0057] Embodiment 3: In vitro safety evaluation of nano toxoid vaccine
[0058] In this example, the in vitro safety of RM-PLs (H1α) was first investigated through hemolysis experiments, and compared with H1α and heat-inactivated H1α. Specific operation: Incubate Hlα, heat-inactivated Hlα (70°C for 30min or 60min), RM-PLs(-), RM-PLs(Hlα) with 2.5% RBC at 37°C, respectively, and centrifuge at 2000g for 5min after a certain period of time , Measure the absorbance value of the supernatant at 540nm and calculate the percentage of hemolysis according to the formula in Example 1. the result shows( image 3 A), Hlα inactivation at 70°C for 30 minutes (70°C 30min Hlα) still has a certain hemolysis effect, and 70°C inactivation for 60 minutes (70°C 60min Hlα) can completely eliminate the hemolysis effect, while RM-PLs (Hlα) has no hemolysis effect, showing Provide better security.
[0059]Next, the in vitro cytotoxicity of RM-PLs (Hlα) was investigated. Specific operation: Extract ...
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