Study On Preparation Of Compound Propolis Nanoemulsion Adjuvant

In order to modify the disadvantages of oil adjuvant, we prepared a new kind of compound propolis nanoemulsion adjuvant. Through stability experiment, security experiment, as well as immune effectiveness experiment, we provided the theoretical basis for the application of compound propolis nanoemulsion adjuvant in preventive veterinary medicine.Part I: The preparation and quality evaluation of Compound propolis nanoemulsion adjuvant. This new form adjuvant was prepared with the method of pseudoternary phase diagrams. RH-40, absolute alcohol, ethyl acetate, propolis, astragalus polysaccharides and distilled water were selected to prepare compound propolis nanoemulsion. Its structural type, appearance, particle size, viscosity, and physical stability were tested respectively. TEM and Nicomp388/Zeta PALS presented the compound propolis nanoemulsion as spherical drops with uniform size distribution and mean diameter of 12.70 nm. PDI is 0.188.The viscosity of compound propolis nanoemulsion was 2s·0.4 mL-1, and good physical stability was proved by stability test. Compound propolis nanoemulsion adjuvant met the requirements of quality of biological products on account of its simple preparation, high stability, low viscosity.Part II: The security evaluation of compound propolis nanoemulsion adjuvant. In this section,we evaluated the safety of compound propolis nanoemulsion adjuvant, through acute toxicity test, eye irritation test, muscle stimulation test, pyrogen test and cytotoxicity test.. The results showed that compound propolis nanoemulsion adjuvant was truly nontoxic, LD50>5000. Compound propolis nanoemulsion adjuvant had no eye irritation and muscle stimulation test, pyrogen inspection conforms to the regulation. Cytotoxicity test showed that there was no significant toxicity to L929 in this adjuvant diluted more than 100 times. Compound propolis nanoemulsion adjuvant met the safety requirements of biological products on account of its high security, low irritation.Part III: The preparation and protective effect of compound propolis nanoemulsion adjuvant inactivated vaccine. In this section, we prepared AIV-H9N2 compound propolis nanoemulsion adjuvant inactivated vaccine, whose quality and security was evaluated. The results showed that the preparation of this kind of vaccine was easy to operate. The prepared vaccine was the type of oil-in-water, transparent and homogeneous, without emulsion breaking phenomenon by centrifuge test at 5000r/min for 30min. The viscosity of vaccine was 2.5s·0.4 mL-1, and the sterility test result met quality standards. The security tests showed that the AIV antigen was completely inactivated, without hemagglutination activity. The vaccine had no adverse reactions to immunized chickens, and protection rate was 100%. This new kind of AIV vaccine was stable, safe and viscosity suitable for injection. So it met requirements of biological products.Part IV: The immune efficacy test of compound propolis nanoemulsion adjuvant. Based on the preliminary studies, humoral immunity and cellular immunity were tested respectively by HI and lymphocyte transformation. The results as follows: When propolis reached the concentration of 15mg/mL, compound propolis nanoemulsion adjuvant inactivated vaccine had a significant immune effect. Compound propolis nanoemulsion adjuvant inactivated vaccine group produced antibody earlier and higher than that of oil adjuvant vaccine group;Compared with white oil adjuvant vaccine group and blank control group, compound propolis nanoemulsion adjuvant inactivated vaccine could stimulate the body to generate stronger cellular immunity. The results implied this new kind of adjuvant had a broad development prospects.