关键词: 日本血吸虫, Sj26gst, mRNA疫苗, 序列优化, 细胞免疫

Abstract:

Objective To evaluate glutathione S-transferase subunit of relative molecular mass (M_r) 26 000 (Sj26gst) mRNA of Schistosoma japonicum and assess its immunogenicity. Methods The Sj26gst coding sequence was optimized by the commonly used human codons according to the Sj26gst coding region sequence (NC_002544.1). The gene was synthesized by adding the 3' and 5' non-coding regions (UTR) of beta-globulin at both ends, and then connected to pcDNA3.1(+) to construct the linear pcDNA3.1/Sj26gst plasmid which was used for transient transcription of Sj26gst mRNA in vitro. The Sj26gst mRNA was transfected into HeLa cells by using liposomes. The HeLa cells were collected to detect the relative expression level of Sj26gst mRNA by quantitative immunofluorescence PCR (qRT-PCR) and examine the expression level of Sj26GST protein by Western blotting at 2, 6, 12, 24, 48 hours after the transfection. Female BALB/c mice were randomly divided into four groups: Sj26gst-mRNA group, pcDNA3.1/Sj26gst group, pcDNA3.1 group and PBS group (6 mice in each group). Mice were immunized three times at two-week interval with 100 μg of Sj26gst mRNA, pcDNA3.1/Sj26gst plasmid, pcDNA3.1 plasmid, or PBS (100 μl), respectively. Blood samples were collected from the tail vein before immunization (0 week) and at 2, 4, 6 and 8 weeks after the first immunization for serum preparation which was used to detect SJ26GST specific IgG antibody levels by ELISA. At 8 weeks post-immunization, the mice spleen tissue was collected aseptically to prepare spleen lymphocyte suspension, which was cultured at 37 ℃ for 48 h, subsequently, 10 μl CCK8 reagent (cell counting kit-8) was added for culturing additional 4 h to determine the spleen cell stimulation index. The levels of cytokines Th1 (TNF-α, INF-γ), Th2 (IL-10, IL-4) and Th17 (IL-17) in the lymphocyte supernatant were assayed by ELISA. One-way analysis of variance was used for inter-group comparisons. Results The optimized coding sequence was 858 bp in length, the GC content was 63.2%, and the free energy change of the secondary structure was -485.6 kcal/mol. The in vitro transcribed Sj26gst mRNA band was a single and non-dispersive band. The RT-PCR results showed that the relative expression level of Sj26gst mRNA in HeLa cells is (6.21 ± 0.83) at 2 h after Sj26gst mRNA transfection, and its peak level was (14.26 ± 1.23) at 6 h, followed by a decrease. The western blotting results showed that Sj26GST protein bands of M_r 26 000 were detected at 6, 12, 24 and 48 hours after Sj26gst mRNA transfection. The ELISA results showed that the A₄₅₀ value of Sj26gst mRNA group, pcDNA3.1/Sj26gst group, pcDNA3.1 group and PBS group were 0.85 ± 0.16, 0.42 ± 0.21, 0.14 ± 0.03 and 0.11 ± 0.02, at eight weeks after the first immunization, respectively. There were statistically significant differences between Sj26gst mRNA group and pcDNA3.1/Sj26gst group, pcDNA3.1/Sj26gst group and pcDNA3.1/Sj26gst group (t = 16.46, 12.35; all P < 0.05). The ELISA results showed that the TNF-α, INF-γ, IL-10, and IL-17 expression levels in the supernatants of splenocytes from Sj26gst mRNA-immunized mice were (756.23 ± 134.35), (598.46 ± 50.47), (713.42 ± 118.25), and (301.45 ± 48.23) pg/ml, respectively. In the PcDNA3.1/Sj26gst group, the corresponding levels were (587.26 ± 32.48), (401.45 ± 46.25), (386.27 ± 23.75) and (175.24 ± 41.23) pg/ml. These levels were higher than those in the pcDNA3.1 group [(19.34 ± 2.01), (35.14 ± 5.25), (32.11 ± 15.20), (19.75 ± 7.21) pg/ml] and the PBS group [(18.75 ± 1.98), (34.12 ± 4.78), (29.36 ± 8.72), (15.34 ± 2.51) pg/ml] (t = 54.59, 19.57, 52.36, 26.47; all P < 0.05). Conclusion The Sj26gst mRNA plasmid with stable expression of Sj26GST was constructed. The Sj26gst mRNA could induce high level of specific antibodies against Sj26GST and stimulate Th1 cellular immune response.

Key words: Schistosoma japonicum, Sj26gst, mRNA vaccine, Sequence optimization, Cellular immune responses