Preparation and application of recombinant argF protein nanoparticles
A nanoparticle and recombinant protein technology, applied in the direction of medical preparations of non-active ingredients, bacterial antigen components, antibacterial drugs, etc., can solve the problems of not having inherent capabilities, reduce tissue loads, and promote BCG immune effects , the effect of increasing IgA levels
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Embodiment 1
[0029] Embodiment 1: the preparation method of recombinant protein argF nanoparticle
[0030] The specific operation steps are as follows:
[0031] (1) Amplify the MB1684 gene of M.bovis, construct the recombinant expression plasmid pProEx-MB1684, transform the recombinant plasmid into BL21 competent cells to obtain recombinant bacteria, induce the recombinant bacteria to express argF with IPTG, and use affinity chromatography to express The argF was purified, dialyzed to desalt, and freeze-dried to obtain the recombinant protein argF;
[0032] (2) Dissolving the lyophilized recombinant protein argF powder in PBS solution as the inner water phase;
[0033] (3) Take an appropriate amount of PLGA, dissolve it in a certain volume of ethyl acetate, and use it as the oil phase for subsequent use;
[0034] (4) Prepare 1% PVA aqueous solution as the external water phase, and 0.5% PVA aqueous solution as the diffusion phase;
[0035] (5) Slowly add the inner aqueous phase into the ...
Embodiment 2
[0039] Example 2: Nanoparticle Characterization and Morphological Observation
[0040] The samples prepared with the ratio of internal water phase and oil phase at 1:9 were characterized, and the particle size and potential were measured using a Malvern Zetasizer nanometer particle size potentiometer. The average particle size of the prepared argF nanoparticles is about 186.6nm, and the potential is -28.8mV. After being measured by an ultraviolet spectrophotometer, the encapsulation efficiency of the nanoparticles was calculated to be 76%. Scanning electron microscope results showed that argF nanoparticles were uniform in size and spherical with a smooth surface ( figure 1 ).
Embodiment 3
[0041] Example 3: Evaluation of Recombinant argF Protein Nanoparticles Enhanced BCG Immune Effect
[0042] The experimental animals were randomly divided into 5 groups, 9 mice in each group, divided into PBS group (no immunization, only challenge), BCG control group (BCG+PBS), BCG+argF group, BCG+PBS-NP group (BCG+blank nanoparticles), BCG+argF-NP group (BCG+argF nanoparticles). Use BCG for the first immunization (approximately 1×10 6 4 weeks later, the recombinant argF protein nanoparticles were boosted for the first time by nasal drops (the dose of argF protein nanoparticles was 75 μg / head), with an interval of 2 weeks, a total of 3 times. Four weeks after the third immunization, 3 mice were randomly selected from each group to detect relevant immune indicators. The rest of the mice were challenged with M.bovis (NTSE-2 strain) by nasal drops (about 1000 CFU / mouse), and samples were taken for follow-up detection after 4 weeks.
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