关键词: 日本血吸虫, 热休克同源蛋白20, 克隆, 表达, 免疫保护效果

Abstract: Objective　To clone and express the heat shock cognate protein 20 (SjHsc20) of Schistosoma japonicum, and to preliminarily investigate its biological characteristics. Methods　The target fragment of the SjHsc20 gene was amplified using PCR assay and cloned into the pET⁃28a(+) expression plasmid to generate the recombinant expression vector pET⁃28a(+)⁃SjHsc20, which was then transformed into Escherichia coli BL21 (DE3) competent cells. The recombinant SjHsc20 (rSjHsc20) protein was induced with isopropyl β⁃D⁃thiogalactopyranoside (IPTG) and purified, and the expression of the rSjHsc20 protein was checked with sodium dodecyl sulfate⁃polyacrylamide gel electrophoresis (SDS⁃PAGE). The immunogenicity of the rSjHsc20 protein was detected using Western blotting, and the transcriptional levels of SjHsc20 were quantified in S. japonicum worms at different developmental stages and in male and female adult worms using real⁃time quantitative PCR (RT⁃qPCR) assay. Thirty female BALB/c mice at ages 6 to 8 weeks were divided into three groups, including the rSjHsc20 immunization group, the PBS control group, and the ISA 206 adjuvant group, of 10 mice in each group. Mice in the rSjHsc20 immunization group were subcutaneously immunized with 20 μg rSjHsc20 on days 1, 15 and 31, and animals in the PBS control group were subcutaneously injected with the same volume of PBS on days 1, 15 and 31, while mice in the ISA 206 adjuvant group were subcutaneously immunized with the same volume of ISA 206 adjuvant on days 1, 15 and 31, respectively. All mice in each group were infected with (40 ± 2) S. japonicum cercariae via the abdomen 14 day following the last immunization. Levels of serum specific IgG and its subtypes IgG1 and IgG2 antibodies against rSjHsc20, and the serum titers of anti⁃rSjHsc20 antibody were detected in mice using indirect enzyme⁃linked immunosorbent assay (ELISA). All mice were sacrifice 42 days post⁃infection, and S. japonicum worms were collected from the hepatic portal vein and counted. The eggs per gram (EPG) , worm burden reductions and egg burden reductions were estimated to evaluate the protective efficacy of the rSjHsc20 protein. Results　The SjHsc20 gene had an open reading frame (ORF) with 756 bp in length and encoded 252 amino acids, and the rSjHsc20 protein had a relative molecular mass of approximately 29 kDa. The rSjHsc20 protein was recognized by the serum of mice infected with S. japonicum and the serum of mice immunized with the rSjHsc20 protein, indicating that rSjHsc20 had a good immunogenicity. There was a significant difference in the transcriptional levels of the SjHsc20 gene among the 7⁃day (1.001 4 ± 0.065 7), 12⁃day (2.268 3 ± 0.129 2), 21⁃day (1.378 5 ± 0.160 4), 28⁃day (1.196 4 ± 0.244 0), 35⁃day (1.646 3 ± 0.226 1), 42⁃day worms of S. japonicum (1.758 0 ± 0.611 1) (F = 38.45, P < 0.000 1), and the transcriptional level of the SjHsc20 gene was higher in the 12⁃day worms than in worms at other developmental stages (all P values < 0.000 1). The serum levels of anti⁃rSjHsc20 IgG antibody were 0.106 6 ± 0.010 7, 0.108 3 ± 0.010 4, and 0.553 2 ± 0.069 1 in the PBS control group, ISA 206 adjuvant group, and rSjHsc20 immunization group following the last immunization, respectively, and the serum levels of IgG1 antibody were 0.137 3 ± 0.054 0, 0.181 1 ± 0.096 8, and 1.765 8 ± 0.221 1, while the levels of IgG2a antibody were 0.280 3 ± 0.197 6, 0.274 0 ± 0.146 3, and 1.560 4 ± 0.106 0, respectively. There were significant differences in the serum levels of anti⁃rSjHsc20 IgG (F = 397.70, P < 0.000 1), IgG1 (F = 401.00, P < 0.000 1) and IgG2a antibodies (F = 229.70, P < 0.000 1) among the three groups, and the serum levels of anti⁃rSjHsc20 IgG, IgG1 and IgG2a antibodies were higher in the rSjHsc20 immunization group than in the PBS control group and the ISA 206 adjuvant group (all P values < 0.000 1). There was a significant difference in the IgG1/IgG2a ratio among the rSjHsc20 immunization group (1.177 2 ± 0.143 6), the PBS control group (0.428 4 ± 0.199 8) and the ISA 206 adjuvant group (0.559 9 ± 0.181 1) (F = 43.97, P < 0.000 1), and the IgG1/IgG2a ratio was > 1 in the rSjHsc20 immunization group, which was higher than in the PBS control group and the ISA 206 adjuvant group (both P values < 0.000 1). The titers of serum anti⁃rSjHsc20 antibody were all above 1∶16 384 in the rSjHsc20 immunization group following immunizations on days 1, 15 and 31, indicating that the rSjHsc20 protein had a strong immunogenicity. The mean worm burdens were (16.60 ± 5.75), (15.80 ± 5.58) worms per mouse and (14.40 ± 5.75) worms per mouse in the PBS control group, the ISA 206 adjuvant group and the rSjHsc20 immunization group 42 days post⁃infection with S. japonicum cercariae (F = 0.50, P > 0.05), and the EPG  were 68 370 ± 22 690, 67 972 ± 19 502, and 41 075 ± 13 251 in the PBS control group, the ISA 206 adjuvant group and the rSjHsc20 immunization group (F = 4.55, P < 0.05), with lower EPG in the PBS control group and the ISA 206 adjuvant group than in the rSjHsc20 immunization group (both P values < 0.05). Immunization with the rSjHsc20 protein resulted in a worm burden reduction of 13.25% and an egg burden reduction of 39.92% relative to the PBS control group.  Conclusions　SjHsc20 is successfully cloned and expressed, and the rSjHsc20 protein induces partial immunoprotective effects in mice, which provides a basis for deciphering the biological functions of SjHsc20 and assessing the potential of SjHsc20 as a vaccine candidate.

Key words: Schistosoma japonicum, Heat shock cognate protein 20, Cloning, Expression, Immunoprotective effect