Molecular and functional characterization of a mortalin-like protein from Schistosoma japonicum (SjMLP/hsp70) as a member of the HSP70 family.
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.
He
Sijie
S
Department of Parasitology, Zhongshan School of Medicine, SunYat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, People's Republic of China.
Yang
Linlin
L
Lv
Zhiyue
Z
Hu
Wei
W
Cao
Jianping
J
Wei
Jie
J
Sun
Xi
X
Yang
Jie
J
Zheng
Huanqin
H
Wu
Zhongdao
Z
D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D015153:Blotting, Western; D019295:Computational Biology; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D018509:DNA, Helminth / Q000235:genetics; D004926:Escherichia coli; D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D050884:HSP72 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012549:Schistosoma japonicum / Q000235:genetics*; D012554:Schistosomiasis japonica / Q000276:immunology / Q000517:prevention & control; D017386:Sequence Homology, Amino Acid; D014611:Vaccination / Q000379:methods
D000909:Antibodies, Helminth; D018076:DNA, Complementary; D018509:DNA, Helminth; D018840:HSP70 Heat-Shock Proteins; D050884:HSP72 Heat-Shock Proteins; D015801:Helminth Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C080352:mortalin