Molecular and functional characterization of a mortalin-like protein from Schistosoma japonicum (SjMLP/hsp70) as a member of the HSP70 family

Parasitol Res. 2010 Sep;107(4):955-66. doi: 10.1007/s00436-010-1960-5. Epub 2010 Jul 3.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Helminth / blood
  • Blotting, Western
  • Computational Biology
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • DNA, Helminth / genetics
  • Escherichia coli
  • Female
  • Gene Expression
  • Gene Expression Profiling
  • HSP70 Heat-Shock Proteins / chemistry
  • HSP70 Heat-Shock Proteins / genetics*
  • HSP70 Heat-Shock Proteins / metabolism*
  • HSP72 Heat-Shock Proteins / chemistry
  • HSP72 Heat-Shock Proteins / genetics*
  • HSP72 Heat-Shock Proteins / metabolism*
  • Helminth Proteins / chemistry
  • Helminth Proteins / genetics*
  • Helminth Proteins / metabolism*
  • Leukocytes, Mononuclear / immunology
  • Mass Spectrometry
  • Mice
  • Mice, Inbred BALB C
  • Molecular Chaperones / genetics
  • Molecular Chaperones / metabolism
  • Molecular Weight
  • Rabbits
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Schistosoma japonicum / genetics*
  • Schistosomiasis japonica / immunology
  • Schistosomiasis japonica / prevention & control
  • Sequence Homology, Amino Acid
  • Vaccination / methods

Substances

  • Antibodies, Helminth
  • DNA, Complementary
  • DNA, Helminth
  • HSP70 Heat-Shock Proteins
  • HSP72 Heat-Shock Proteins
  • Helminth Proteins
  • Molecular Chaperones
  • Recombinant Proteins
  • mortalin