*2.4. Animal Experiment*

The animal experiments were conducted in Beijing Animal Experimental Center. The Animal Ethics Committee of IAR, CAAS (Beijing, China) provided approval for the animal experimentation. The registration number is CAAS-IAR-ER0046. Female BALB/c mice (6-week-old, 16–20 g) were kept in cages, housed in a 12-h light–dark cycle at 20–24 ◦C and a relative humidity of 50 ± 5%, and supplied with filtered pathogen-free air, standard AIN-93 laboratory diet and sterile water. After acclimation for one week, all mice were randomly divided into five groups (*n* = seven per group), and named as CK, OVA, BP, 2E-BP and 3E-BP. The mice administration procedure is shown in Figure 1A. Briefly, mice in all groups were given a standard diet and water. For the CK group, mice were intraperitoneally injected with a saline solution every seven days for 28 days; but for the OVA group (as a positive control), mice were injected intraperitoneally with 0.2 mL of 0.1 mg/mL OVA solution (containing 1% Alum Adjuvant) every seven days for 21 days, and finally injected with 0.2 mL of 0.5 mg/mL OVA solution (containing 1% Alum Adjuvant) on the 28th day; for BP, 2E-BP and 3E-BP groups, mice were injected intraperitoneally with 0.2 mL of 0.5 mg/mL BP, 2E-BP and 3E-BP solution (containing 1% Alum Adjuvant) every seven days for 21 days, respectively, and finally injected with 0.2 mL of 2.5 mg/mL BP, 2E-BP and 3E-BP solution (containing 1% Alum Adjuvant) on the 28th day, respectively. The frequency of scratching behavior was observed and recorded 15 min after final injection. Mice were sacrificed after intraperitoneal injection with an excitation dose of samples for 30 min on the 28th day. The blood was extracted from eyes, and then centrifuged at 3000 rpm for 10 min for serum collection.

**Figure 1.** BALB/c mice administration schedule. (**A**) Scratching frequency of mice in different treatment groups. (**B**) Different letters indicate a significant difference among different groups (*p* < 0.05).
