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

• Published in: Journal of helminthology 2020-01, Vol.94, p.e118-e118, Article e118
• Main Authors: Hang, J X, He, L, Abuzeid, A M I, Huang, Y, Liu, Y Q, Yan, X X, Zhao, Q, Li, X, Liu, J M, Li, G Q
• Format: Article
• Language: English
• Subjects: Amplification; Ancylostoma - genetics; Ancylostoma - immunology; Ancylostoma - metabolism; Ancylostomiasis; Anemia; Animals; Antibodies; Antibodies, Helminth; Antibody response; Antigens, Helminth - genetics; Antigens, Helminth - metabolism; Binding sites; Biological evolution; Cloning; Cloning, Molecular; Complementary DNA; Deoxyribonucleic acid; Disease; Disease control; DNA; DNA, Helminth - genetics; DNA, Helminth - isolation & purification; Drug resistance; E coli; ELISA; Enzyme-linked immunosorbent assay; Enzymes; Epidermis; Escherichia coli - genetics; Genetic analysis; Glutathione; Glutathione transferase; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Homology; IgG antibody; Immunization; Immunofluorescence; Immunoglobulin G; Immunoglobulins; Immunohistochemistry; Infections; Intestine; Intestines; Laboratory animals; Lipid peroxidation; Localization; Muscles; Nematodes; Nucleotide sequence; Parasites; PCR; Phylogeny; Polymerase chain reaction; Primers; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombinants; Reverse transcription; Serum; Skin; Survival; Tissue; Transcription; Transferases; Transformation, Bacterial; Tropical diseases; Vaccine development; Vaccines; Worms
• ISSN: 0022-149X; 1475-2697
• DOI: 10.1017/S0022149X20000012

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.