2.2.1Preparation and characterization of W541 DNA vaccine and its corresponding recombinant protein W540.
According to the genetic code rules, the amino acid sequence of the W541 vaccine protein was translated into a DNA sequence. The DNA sequence was then codon-optimized to generate sequences specifically suited for eukaryotic and prokaryotic expression. The optimized eukaryotic expression DNA sequence and the pVAX1 vector plasmid were digested with restriction enzymes NheI and EcoRI, respectively. The two fragments were ligated using DNA ligase to construct the recombinant plasmid. The recombinant plasmid was then transformed into Escherichia coli (E. coli ) DH5α competent cells. Subsequently, the transformed E. coli DH5α cells were cultured in the LB medium. After culturing, the W541 recombinant plasmid was isolated and purified using the plasmid extraction kit (Qiagen, Germany). Similarly, the optimized prokaryotic expression DNA sequence and the pET28a vector plasmid were digested with restriction enzymes NheI and EcoRI, respectively. Then, the obtained fragments were ligated using DNA ligase to construct the W540 recombinant protein plasmid and transformed into E. coliBL21(DE3) competent cells. The transformed E.coli BL21(DE3) cells were cultured on LB medium and induced by 0.1 mM IPTG at 37℃ for 3 hours before purifying the recombinant fusion protein(62). The W541 recombinant plasmid was identified by sequencing and enzyme digestion. The W540 recombinant plasmid was identified by sequencing, and the protein expression of transformed E.coli BL21(DE3)was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (63).
2.2.2 Experimental animals
Female BALB/c mice aged 56-62 days and weighing 18-20g were obtained from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). The animal experiments were conducted following the Regulations on Management of Experimental Animals formulated by the Ministry of Science and Technology of China. The Animal Ethics Committee of the Eighth Medical Center of PLA General Hospital has approved the experimental plan.
2.2.3 Immunization of the Animals
Mice were divided into three groups (8 mice per group) and treated as follows: 1) Control group: each mouse was injected intramuscularly with 100 μl of saline; 2) pVAX1 group: each mouse was injected intramuscularly with 100 μg pVAX1 plasmid diluted in 100 μl saline; 3) Vaccine group: each mouse was injected intramuscularly with 100 μg W541 DNA vaccine diluted in 100 μl saline (32). Mice received injections every two weeks for a total of 3 injections. At five weeks after the last immunization, the mice were euthanized. Blood samples were collected in heparin lithium anticoagulant tubes, and then the plasma was separated. The spleens of the mice were also harvested.
2.2.4 Detection of specific antibodies against W540
The blood sample of each mouse was separated from the plasma and stored at -20℃. The enzyme-linked immunosorbent assay (ELISA) was employed to individually detect specific antibodies IgG and its subtypes IgG1 and IgG2a against W540 protein in the plasma samples obtained from all 24 mice in 3 groups. The method is briefly described as follows: The ELISA plate (Costar, China) was coated with 100 μl of 10 μg/ml W540 protein in the coating buffer (0.05M NaHCO3, pH 9.6) and incubated overnight at 4℃. The plate was then washed with PBS-T (1×Phosphate-Buffered Saline, pH7.4, containing 0.05% Tween-20) and blocked with 200 μl/well of 2% (w/v) ovalbumin (Coolaber, China) in PBS buffer and incubated for 3 hours at 37℃. Plasma samples were diluted 1:100 in assay buffer (1% w/v BSA in PBS-T) and added in duplicate at 100 μl/well, followed by incubation for 2 hours at 37℃. The coating buffer without W540 protein was added to each plate as a negative control. Goat anti-mouse IgG, IgG1, and IgG2a conjugated to horseradish peroxidase (HRP) (Abcam, England) diluted 1:10,000 in assay buffer was added at 100 μl/well and incubated for 1 hour at 37℃. After washing, 100 μl of 3,3’,5,5’-Tetramethylbenzidine(TMB) substrate (BD, America) was added to each well, and plates were incubated in the dark at room temperature for 5 minutes. The enzymatic reaction was stopped by adding 50 μl of 1M H2SO4, and then the absorbance at 450nm was measured using a microplate reader (Thermo, America).
2.2.5 Detect the number of mouse splenocytes secreting IFN-γ by enzyme-linked Immunospot assay (ELISPOT)
Equal amounts of mice spleen cells from the same group were mixed to reduce individual variations, and 3 independent experiments were subsequently conducted with the Mouse IFN-γ ELISpotPLUS (HRP) assay kit (MABTECH AB, Sweden)to assess the immune response of mouse splenocytes to the W540 protein. The detailed process follows: 3×105mixed cells were seeded in each well of the filter membrane plate pre-packaged with IFN-γ capturing antibody (MABTECH AB, Sweden). The negative control wells were added with RPMI 1640 complete medium, while the positive control wells were added phytohemagglutinin (PHA) (Sigma, USA) at a final concentration of 20mg/ml. Following the kit’s instructions, the number of splenocytes secreting IFN-γ was measured after stimulation with W540 protein at a concentration of 30µg/ml in a CO2 incubator at 37℃ for 20 hours. The number of spots in each well was detected using CTL ImmunoSpot®S5 Micro equipment (Cellular Technology, America).
2.2.6 Cytokine analysis
Spleen cell suspensions from each group of mice were mixed in equal proportion to reduce individual variations, and then 3 repeated experiments were conducted. The mixed spleen cell concentration was adjusted to 3×106 cells/ml. After stimulation with W540 protein at a final concentration of 30µg/ml for 24h, the supernatant from spleen cell cultures was subjected to analyze the expression levels of cytokine IFN-γ, IL-2, IL-4, IL-6, IL-10, and IL-17A using the CBA assay kit (BD Biosciences, USA) following the instructions provided. The measurements were performed in triplicate.
2.2.7 Statistical analysis
The antibody detection, ELISPOT assay, and cytokine detection data were analyzed using GraphPad Prism 8 software. Quantitative data of normal distribution were analyzed using one-way analysis of variance (ANOVA), and pairwise comparisons were conducted using Tukey s multiple comparisons test. The results were presented as mean±standard error, and a p-value of less than 0.05 was considered a statistically significant difference.
3 Results