日本血吸虫热休克蛋白40 kDa免疫学功能研究  

Study on immunological functions of heat shock protein 40 kDa of Schistosoma japonicum

作　　者：余艳雄 李莎莎[1] 朱继峰[1] 陈晓军[1] 徐志鹏[1] 李娅琳[1] 周莎[1] 苏川[1] YU Yan-Xiong;LI Sha-Sha;ZHU Ji-Feng;CHEN Xiao-Jun;XU Zhi-Peng;LI Ya-Lin;ZHOU Sha;SU Chuan(Department of Pathogenic Biology,Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]南京医科大学病原生物学系,南京210029

出　　处：《中国血吸虫病防治杂志》2020年第3期262-267,272,共7页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金青年基金(81601789);江苏省自然科学基金优秀青年基金(BK20170105)。

摘　　要：目的探索日本血吸虫热休克蛋白40 k Da(Sj HSP40)免疫学功能。方法采用生物信息学方法分析Sj HSP40蛋白序列同源性,并预测其B细胞及T细胞表位。应用PCR技术扩增全长Sj HSP40基因,将其克隆入原核表达质粒p GEX-6P-1,再转入大肠埃希菌BL21中;以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,再经亲和柱分离纯化获得谷胱甘肽巯基转移酶(GST)-Sj HSP40融合蛋白,分别采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western blotting进行鉴定。以GST-Sj HSP40免疫BALB/c小鼠后,以酶联免疫吸附试验(ELISA)检测小鼠血清中抗Sj HSP40特异性Ig G抗体及其亚型Ig G1、Ig G2a抗体,采用流式检测术观察Sj HSP40对CD4+T细胞亚群分化的影响。结果 Sj HSP40含有7个潜在B细胞表位及多个T细胞表位(CTL表位和Th表位)。成功构建了原核表达重组质粒p GEX-6P-1-Sj HSP40,并通过诱导表达和蛋白纯化获得GST-Sj HSP40融合蛋白。与其他组相比,GST-Sj HSP40免疫小鼠血清中抗Sj HSP40特异性Ig G抗体及其亚型Ig G1和Ig G2a水平均显著升高,但Sj HSP40对CD4+T淋巴细胞分化无显著影响。结论 Sj HSP40可诱导小鼠产生特异性体液免疫应答,但不影响小鼠体内各类Th免疫反应平衡,可作为潜在疫苗候选分子。Objective To investigate the immunological functions of heat shock protein 40 kDa of Schistosoma japonicum(Sj HSP40).Methods The homology of the Sj HSP40 protein sequence was analyzed and the B and T cell epitopes of Sj HSP40 were predicted using bioinformatics tools. The full-length Sj HSP40 gene was amplified using a PCR assay, and cloned into theprokaryotic expression vector p GEX-6 P-1, which was transformed into Escherichia coli BL-21. The protein expression was in-duced with isopropyl β-D-thiogalactoside(IPDG), and then, the recombinant protein was purified with glutathione-sepharose 4 Bresin to yield the fusion protein GST-Sj HSP40, which was checked with SDS-PAGE and Western blotting. Following immuniza-tion with GST-Sj HSP40, the serum levels of anti-Sj HSP40 Ig G antibody and Ig G1 and Ig G2 a subtypes were detected in BALB/cmice using ELISA. In addition, the effect of Sj HSP40 on CD4+T-cell subset differentiation was examined using flow cytometry.Results Sj HSP40 contained 7 potential B cell epitopes and multiple T cell epitopes(CTL epitopes and Th epitopes). The pro-karyotic expression plasmid p GEX-6 p-1-Sj SHP40 was successfully constructed, and the fusion protein GST-Sj HSP40 was ob-tained following IPDG induction and protein purification. Significantly higher serum levels of anti-Sj HSP40 Ig G, Ig G1 and Ig G2 aantibodies were detected in mice immunized with GST-Sj HSP40 than in other groups;however, Sj HSP40 showed no remarkableeffects on CD4+T-cell subset differentiation.Conclusions Sj HSP40 may induce specific humoral immune responses in mice;however, it does not affect the balance of Th immune responses. It is suggested that Sj HSP40 may be a potential vaccine candi-date.

关 键 词：日本血吸虫 热休克蛋白40 kDa 免疫学功能 体液免疫 

分 类 号：R383.24[医药卫生—医学寄生虫学]