日本血吸虫糖基化磷脂酰肌醇锚定蛋白的鉴定

Identification of Glycosylphosphatidylinositol-anchored Protein from Schistosoma japonicum

目的鉴定日本血吸虫的糖基化磷脂酰肌醇锚定蛋白(GPI-AP)。方法根据曼氏血吸虫的糖基化磷脂酰肌醇(GPI)锚定蛋白Sm200的编码基因(GenBank登录号为XM_002569560.1),运用生物信息学方法寻找日本血吸虫的同源基因,分析后选取基因蛋白质编码区(CDS)的部分基因序列(SjGPIs,长约933 bp)进行PCR扩增,并克隆入原核表达载体pET-28a(+),重组质粒转化大肠埃希菌(E.coli)BL21(DE3)感受态细胞,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,用镍柱Ni-NTA亲和层析纯化重组肽段SjGPIs。用纯化的重组肽段SjGPIs免疫新西兰大耳兔,以制备的重组肽段抗血清检测日本血吸虫GPI锚定蛋白。用磷脂酰肌醇特异性磷脂酶C(PI-PLC)鉴定检测到的蛋白在日本血吸虫虫体上的锚定方式。检测日本血吸虫感染小鼠的白细胞,确定其是否吞噬GPI锚定蛋白。结果日本血吸虫的基因组中存在与曼氏血吸虫GPI锚定蛋白Sm200基因的同源基因序列,经比对拼接后获得3 495 bp含完整编码蛋白C末端的基因编码序列。以所选基因序列进行肽段原核表达,获得重组质粒pET-28a(+)-SjGPIs。通过对蛋白C末端序列分析、经蛋白质印迹(Wes-tern blotting)分析和PI-PLC酶切验证,发现日本血吸虫被膜存在以GPI形式锚定的蛋白,相对分子质量约为Mr200 000,命名为SjGPI200。感染日本血吸虫小鼠的白细胞中可检测到完整的SjGPI200蛋白。结论日本血吸虫存在锚定蛋白SjGPI200,并以GPI形式锚定于虫体被膜上。

Objective To identify glycosylphosphatidylinositol （GPI） anchored protein of Schistosoma japonicum. Methods Based on the gene sequence of Schistosoma mansoni GPI anchored protein Sm200（GenBank Assess No：XM_002569560.1）, bioinformatics analysis was performed to find out its homologous gene sequence in S. japonicum, then a selected partial coding sequence （SjGPIs, about 933 bp） from the homologous gene sequence were amplified, and cloned into PET-28a（＋） vector. The recombinant plasmid pET-28a（＋）-SjGPIs were transformed into E. coli Top10 cells and induced with IPTG for protein expression. The recombinant protein SjGPIs was purified with Ni-NTA resin, and the purified recombinant SjGPIs protein was used as antigen to prepare antiserum in New Zealand rabbit. The antiserum was used to detect S. japonicum GPI-anchored protein. To identify a GPI-anchored protein, the detected protein were identified by phosphatidylinositol-specific phospholipase C （PI-PLC） digestion. White blood cells from S. japonicum-infected mice was examined whether they endocytosed GPI-anchored proteins by Western blotting. Results The homologous gene sequence of S. mansoni GPI Sm200 gene was found in S. japonicum genome. A 3 495 bp coding sequence was obtained, containing the complete C-terminal sequence. The selected gene sequence （SjGPIs） were amplified and the recombinant plasmid pET-28a（＋）-SjGPIs was established. According to the analysis of C-terminal sequence, Western blotting and enzyme digestion of PI-PLC, a GPI-anchored protein was present in S. japonicum tegument （about Mr 200 000）, named SjGPI200. The protein was detected in white blood cells of infected mice. Conclusion SjGPI200 protein exists in S. japonicum, and anchored to parasite tegument via GPI.