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

摘要： 目的　克隆、表达日本血吸虫（Schistosoma japonicum，Sj）热休克同源蛋白20（heat shock cognate protein 20，Hsc20）（SjHsc20），并对其生物学特性进行初步研究。方法　采用PCR法扩增SjHsc20基因目的片段，构建重组表达载体pET⁃28a（+）⁃SjHsc20，并将其转化入大肠埃希菌BL21（DE3）。采用异丙基⁃β⁃D⁃硫代半乳糖苷（isopropyl β⁃D⁃thiogalactopyranoside，IPTG）诱导表达并纯化重组SjHsc20（rSjHsc20）蛋白，采用十二烷基硫酸钠⁃聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate⁃polyacrylamide gel electrophoresis，SDS⁃PAGE）验证重组蛋白表达，采用Western blotting检测重组蛋白免疫原性。采用实时荧光定量PCR（real⁃time quantitative reverse transcription polymerase chain reaction， RT⁃qPCR）法检测SjHsc20基因在不同发育期虫体及不同性别成虫中的转录水平。30只雌性6～8周龄BALB/c小鼠随机分为rSjHsc20免疫组、PBS对照组、ISA 206佐剂组，每组10只。在实验第1、15、31天，rSjHsc20免疫组、PBS对照组、ISA 206佐剂组小鼠分别皮下免疫注射20 μg rSjHsc20重组蛋白及等体积PBS和ISA 206佐剂，最后1次免疫14 d后，各组小鼠经腹部贴片法感染日本血吸虫尾蚴（40 ± 2）条。采用间接酶联免疫吸附试验（enzyme⁃linked immunosorbent assay，ELISA）检测各组小鼠血清抗rSjHsc20特异性IgG及其亚型IgG1、IgG2a抗体水平，以及血清中抗rSjHsc20抗体滴度，并计算IgG1/IgG2a比值。感染42 d后，剖杀各组小鼠，采用肝门静脉灌注法收集虫体并计数，计算每克肝脏虫卵数（eggs per gram，EPG）以及rSjHsc20免疫组相对于PBS对照组的减虫率和减卵率，以评估rSjHsc20蛋白免疫保护效果。结果　SjHsc20基因开放阅读框长756 bp、编码252个氨基酸，rSjHsc20蛋白相对分子质量约为29 kDa。rSjHsc20可被感染日本血吸虫的小鼠血清以及免疫该蛋白后获得的小鼠血清识别。7、12、21、28、35、42 d虫体SjHsc20基因相对转录水平分别为1.001 4 ± 0.065 7、2.268 3 ± 0.129 2、1.378 5 ± 0.160 4、1.196 4 ± 0.244 0、1.646 3 ± 0.226 1、1.758 0 ± 0.611 1，差异有统计学意义（F = 38.45，P < 0.000 1），其中12 d虫体内SjHsc20基因转录水平高于其他时期虫体（P均< 0.05）。第3次免疫后，PBS对照组、ISA 206佐剂组和rSjHsc20免疫组小鼠血清IgG抗体水平分别为0.106 6 ± 0.010 7、0.108 3 ± 0.010 4、0.553 2 ± 0.069 1，IgG1抗体水平分别为0.137 3 ± 0.054 0、0.181 1 ± 0.096 8、1.765 8 ± 0.221 1，IgG2a抗体水平分别为0.280 3 ± 0.197 6、0.274 0 ± 0.146 3、1.560 4 ± 0.106 0，差异均有统计学意义（F = 397.70、401.00和229.70，P均< 0.000 1）；两两比较结果显示，rSjHsc20免疫组小鼠血清IgG、IgG1和IgG2特异性抗体水平均高于其他两组（P 均< 0.000 1）。3组小鼠血清IgG1/IgG2a比值分别为0.428 4 ± 0.199 8、0.559 9 ± 0.181 1、1.177 2 ± 0.143 6，差异有统计学意义（F = 43.97，P < 0.000 1）；rSjHsc20免疫组IgG1/IgG2a比值> 1，且高于PBS对照组和ISA 206佐剂组（P 均< 0.001）。3次免疫后，rSjHsc20免疫组小鼠血清抗体滴度均在1∶16 384 以上，表明rSjHsc20具有较强免疫原性。在尾蚴攻击42 d后，PBS对照组、ISA 206佐剂组、rSjHsc20免疫组小鼠平均虫荷数分别为（16.60 ± 5.75）、（15.80 ± 5.58）、（14.40 ± 5.75）条/鼠，差异无统计学意义（F = 0.50，P > 0.05）；3组小鼠EPG分别为68 370 ± 22 690、67 972 ± 19 502、41 075 ± 13 251，差异有统计学意义（F = 4.55，P < 0.05）；两两比较结果显示，PBS对照组和ISA 206佐剂组EPG均低于rSjHsc20免疫组（P均 < 0.05）。rSjHsc20免疫组小鼠减虫率和减卵率分别为13.25%和39.92%。结论　成功克隆、表达了SjHsc20基因，rSjHsc20蛋白可诱导小鼠产生一定的免疫保护效果。上述结果为后续研究SjHsc20生物学功能及评估其作为疫苗候选分子的潜能奠定了基础。

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