Molecular and functional characterization of a mortalin-like protein from Schistosoma japonicum (SjMLP/hsp70) as a member of the HSP70 family
Molecular and functional characterization of a mortalin-like protein from Schistosoma japonicum (SjMLP/hsp70) as a member of the HSP70 family
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DOI：
10.1007/s00436-010-1960-5
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发表时间：
2010-09-01
影响因子：
2
通讯作者：
Wu, Zhongdao
中科院分区：
文献类型：
--
作者：
He, Sijie;Yang, Linlin;Wu, Zhongdao
血吸虫是血吸虫病的病原体。70 kDa热休克蛋白(HSP70)被认为是主要的热休克蛋白家族，在寄生虫的发育和致病过程中起着关键的调节作用。通过生物信息学分析，获得了一个编码653个氨基酸(XP_002581385.1)的开放阅读框，属于血吸虫HSP70蛋白家族，相对分子质量为71.49 kDa。由于该序列与已发表的智人HSP70全长蛋白有77%的同源性，故将其命名为血吸虫死亡蛋白样蛋白(MLP/Hsp70)。在这里，我们报道了日本血吸虫SjMLP/HSP70作为HSP70家族成员的分子和功能特征。利用聚合酶链式反应(PCR)从日本血吸虫成虫cDNA文库中扩增出SjMLP/HSP70全长编码序列，并将其亚克隆到pET28a表达载体中。纯化的重组蛋白rSjMLP/HSP70经质谱鉴定为70 kDa HSP家族成员，可被日本血吸虫感染的小鼠血清识别。逆转录聚合酶链式反应(RT-PCR)和免疫印迹分析表明，SjMLP/HSP70在日本血吸虫的卵、尾蚴、血吸虫和成虫中广泛表达。耐热性实验表明，rSjMLP/HSP70能保护大肠杆菌细胞免受热损伤。全长SjMLP/HSP70证明了这种伴侣样活性。间接酶联免疫吸附试验检测特异性抗体水平，ELISpot法检测小鼠脾细胞分泌干扰素-γ，提示SjMLP/HSP70免疫小鼠可诱导Th1型偏向免疫应答。攻击保护实验表明，SjGST DNA疫苗与SjMLP/HSP70联合免疫可使小鼠肠道组织的虫卵负担分别减少31.31%和58.59%。我们的结果提示SjMLP/HSP70具有潜在的佐剂功能，有可能成为血吸虫病疫苗的候选分子，这是该分子的表达和初步特性分析的首次报道。
Schistosomes are the causative agent of schistosomiasis. The 70-kDa heat-shock proteins (HSP70) are considered the predominant HSP family and play a key regulatory role in parasite development and pathogenesis. Based on the published sequences in Genbank/EMBL, an open-reading frame (ORF) encoding 653 amino acids (XP_002581385.1) and belonging to the Schistosoma HSP70 protein family with a molecular weight of 71.49 kDa was identified by bioinformatic analysis. Since the sequence shared 77% identity with the published full-length Homo sapiens HSP70 protein, it was named Schistosoma mortalin-like protein (MLP/Hsp70). Here, we report the molecular and functional characterization of the Schistosoma japonicum SjMLP/hsp70 as a member of the HSP70 family. The complete SjMLP/hsp70 coding sequence was amplified from a S. japonicum adult worm cDNA library by polymerase chain reaction (PCR) and subcloned into the pET28a expression vector. The purified recombinant protein, rSjMLP/hsp70, was identified as a member of 70-kDa HSP family by mass spectrometry and could be recognized by the S. japonicum-infected mouse serum. Reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting analysis revealed that SjMLP/hsp70 was widely expressed in the eggs, cercariae, schistosomula, and adult worms of S. japonicum. A thermotolerance assay showed that rSjMLP/hsp70 could protect Escherichia coli cells from heat damage. This chaperone-like activity was demonstrated by full-length SjMLP/hsp70. The detection of specific antibody levels by indirect enzyme-linked immunosorbent assay and IFN-gamma secretion of splenocytes by ELISpot assay suggested that mice immunized with SjMLP/hsp70 were able to elicit Th1-type bias immune response. The challenge-protective experiment showed that DNA vaccine of SjGST combined with SjMLP/hsp70 could induce a 31.31% reduction of worm burden and 58.59% reduction of egg burden in intestinal tissue of immunized mice. Our results imply that SjMLP/hsp70 has a potential adjuvant function and might be a vaccine candidate for schistosomiaisis, which is the first report of the expression and preliminary characterization analysis of this molecule.