Molecular cloning and characterization of a HSP70 gene from Schistosoma japonicum

Abstract

Schistosoma japonicum is the pathogen responsible for schistosomiasis japonica, one of the major infectious diseases targeted for prevention nationally in China. Expression of heat shock proteins (HSPs) following stress plays a very important biological role in many organisms including S. japonicum. Among the HSP family, the 70-kDa HSPs are most responsible for intracellular chaperone and extracellular immunoregulatory functions. Based on the published sequences in GenBank/EMBL (AF044412.1), open reading frame belonging to HSP70 protein corresponds to a full-length cDNA containing an open reading frame of 1,947 bp encoded of 648 amino acids was identified as HSP70 from schistosome. In this study, the coding region that we named rSj648/hsp70 was amplified from S. japonicum adult worm cDNA library, and the recombinant protein was expressed in vector pET32a(+) and purified using a Ni-NTA purification system. The target protein rSj648/hsp70 was determined by matrix-assisted laser desorption/ionization mass spectrometer after thrombin digestion and dialysis. Reverse transcriptase polymerase chain reaction and Western blotting analysis confirmed that Sj648/hsp70 could be expressed in the eggs, normal cercariae, ultraviolet-attenuated cercariae (UVAC), and adult worms of S. japonicum. Real-time quantitative PCR analysis indicated that Sj648/hsp70 was expressed significantly higher in eggs than that in cercariae and adult worms, and the expression in UVAC was higher than that in normal cercariae. A thermotolerance assay showed that rSj648/hsp70 could protect Escherichia coli cells from heat damage. The detection of specific antibody levels by indirect enzyme-linked immunosorbent assay demonstrated that mice immunized with rSj648/hsp70 induced higher level of specific anti-rSj648/hsp70 IgG1 compared with those vaccinated with adjuvant alone, indicating that rSj648/hsp70 was able to elicit Th2-type bias immune response. Our results suggest that Sj648/hsp70 might be an important molecule in parasite-host interaction and display potential roles in mice immunoregulation system.