Molecular characterization and tissue localization of glutathione S -transferase from adult Ancylostoma ceylanicum

Glutathione S -transferases (GSTs) are a detoxifying enzyme family that is essential for parasite blood-feeding and survival, and represent potential targets for hookworm vaccine development. Multiple GST-encoding complementary DNAs (cDNAs) have been cloned from Ancylostoma caninum and Necator americanus , but there are no reports about the cloning of this enzyme from Ancylostoma ceylanicum , the animal-derived zoonotic hookworm. To study the molecular nature and tissue localization of GST of A. ceylanicum ( Ace -GST), we designed primers based on the GST gene sequence of A. ceylanicum in GenBank, amplified the Ace -GST cDNA by reverse transcription polymerase chain reaction, and analysed its homology and genetic evolution relationship. The amplified product was cloned into the pET-32a vector and transformed into Escherichia coli BL21 (DE3) for expression. To prepare anti-GST polyclonal antibodies, the recombinant protein was purified and used to immunize Kunming mice. The level of immunoglobulin G (IgG) antibody in the serum of immunized mice was detected by indirect enzyme-linked immunosorbent assay, and the Ace -GST localization in adult worm was determined using the immunofluorescence method. The results showed that the full-length cDNA encoding Ace -GST was 468 bp, which had the highest homology with Ac -GST-1 (60.1%) and clustered into one branch ( v -class) with Ac -GST-1 and Na -GST-1 in a phylogenetic tree. Mice immunized with recombinant Ace -GST showed specific IgG antibody response. Immunolocalization revealed that natural Ace -GST is mainly located in the epidermis, muscle and intestine of the adult. These results may lay a foundation for further studies on the biological function of Ace -GST.