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OBJECTIVE To optimize the formulation process of liposomes, construct a complex adjuvant liposome peptide vaccine, and evaluate its immune effect in mice. METHODS Taking particle size distribution and polydispersity index as evaluation indicators, the formulation process was optimized through single factor experiments. The physical properties were investigated using a laser particle size analyzer, and the microstructure was observed using transmission electron microscopy. The encapsulation efficiency was calculated using BCA protein concentration assay, and the stability was investigated by storing at 4 and 25 ℃ for 0, 90, and 180 d. Three composite adjuvant peptide vaccines were constructed by combining it with squalene, monophosphoryl lipid A(MPLA), and QS-21. Then, BALB/c mice were randomly divided into a blank control group and an experimental group. The experimental group was subcutaneously injected with free peptide vaccine and three kinds of liposomal peptide vaccine, respectively. The samples were taken 14 d after the second immunization. ELISA was used to detect the specific IgG antibody and its subtype titer in mouse serum, and flow cytometry was used to detect the spleen lymphocyte typing, to evaluate the immune effects of different peptide vaccine formulations. RESULTS The optimal preparation conditions obtained from single factor experiments were as follows: membrane material ratio of 5∶1, ultrasonic power of 30 W, ultrasonic frequency of 40 times, and high-pressure homogenization time of 6 min. The average particle size of the liposomes was reduced from 329.7 nm to 132.0 nm, with a polydispersity index of 17.8% and an encapsulation efficiency of 76.9%. The morphology under transmission electron microscopy was regular and approximately spherical, and the stability was good after storage at 4 ℃ for 180 d. The results of the in vivo immune experiment in mice showed that the liposomal peptide vaccine could produce high titer IgG and IgG2a antibodies, up to 6.4×104 and 3.2×104, respectively; upregulate the proportion of CD4+T and CD8+T cells in lymphocytes (P<0.05), and enhance cellular immunity in mice. CONCLUSION The physicochemical properties of the optimized formulation process are stable and good, and the polypeptide vaccine formulated with adjuvant can enhance humoral and cellular immunity in mice。 It is a potential carrier for the delivery of polypeptide antigen immunization.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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