Fig. 1

Construction and design of CD40L multi-epitope fusion trimer protein"

Table 1

Immunization grouping of guinea pigs"

Table 2

Primers used for cytokine gene expression assay by real-time RT-PCR"

Fig. 2

>Optimization of pig 5B CD40L protein expression condition A. Optimization of protein expression temperature (M. Protein molecular weight standard; 1.2. Before and after induction at 16 ℃; 3, 4. Before and after induction at 37 ℃); B. Soluble expression of the target protein (M. Protein molecular weight standard; 1.2. Precipitation and supernatant after ultrasound at 37 ℃; 3, 4. Precipitation and supernatant after ultrasonic treatment at 16 ℃; C. Optimization of inducer IPTG concentration (M. Protein molecular weight standard; 1-7.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 mmol·L-1; D. Optimization of bacterial density OD600 nm value (M. Protein molecular weight standard; The values of 1 to 4.OD600 nm are 1.1, 0.9, 0.7, and 0.5 respectively"

Fig. 3

5B CD40L protein purification and identification A. SDS-PAGE; B. Western blot; M. Protein molecular weight standard; 1.5B CD40L"

Fig. 4

Assays of purified recombinant 5B CD40L proteins by Native-PAGE"

Fig. 5

Activity detection of FITC-5B CD40L protein"

Fig. 6

Gate-setting strategy of B cells and detection of 5B CD40L bioactivity in vitro A. B-cell sorting strategy (using the dual parameter characteristics of forward scattering angle (FSC-A) and side scattering angle (SSC-A) to conduct an initial screening of the lymphocyte population, and further identifying B-cell subgroups through anti-CD21 antibody labeling); B. The flow cytometry scatter plot presents the co-expression characteristics of the co-stimulatory molecules CD80 and CD86 on the surface of CD21+ B lymphocytes in the 5B CD40L treatment group; C. Quantitative analysis of five independent experiments showed (n=5), there was a dose-dependent difference in the average fluorescence intensity of CD80/CD86 in CD21+ B lymphocytes of the test group, and the data are presented as the ${\bar x}$±s. ns. No difference, *. P < 0.05, **. P < 0.01, ***. P < 0.001, ****. P < 0.000 1, the same as below"

Fig. 7

Identification and characterization of the VLPs A. Identification by 10% SDS-PAGE; B. Identification by Western blot with 12% SDS-PAGE; C. Characterization by TEM in 200 nm scale (80 k V); M. Protein Marker; 1. VLPs"

Fig. 8

Detection of the neutralization antibody A. Curve of neutralizing antibody titer with a time line on the X axis; B. Neutralizing antibody level comparison at 14 and 35 dpi"

Fig. 9

Detection of the lymphocyte stimulation"

Fig. 10

Cytokine genes transcription evaluation by real-time RT PCR"