Schistosoma haematobium protein array features
Information on the Schistosoma haematobium antigen features that were printed on a proteome array for subsequent probing with serum and urine antibodies.
Steps to reproduce
Schistosoma haematobium protein array feature selection and construction. The primary criterion for the selection of proteins to be included on the S. haematobium protein array was the presence of the protein in surface and secreted proteomes of the parasite as these molecules are likely to be exposed to the host upon infection, and their cognate antibodies contained within the immune repertoire of infected individuals. Accordingly, proteins present in the adult S. haematobium tegument, soluble excretory/secretory products (ES) and Extracellular Vesicles (EVs), ES from the egg stage,1,4 and S. haematobium orthologues of proteins present in the Schistosoma mansoni schistosomula tegument proteome (650 proteins in total) were selected for the array. The remaining ~350 proteins (for a ~1,000 feature array) consisted of S. haematobium orthologues of select proteins featured on the next generation S. mansoni proteome array. Open reading frames (minus predicted signal peptides and flanked by 20 bp sequences corresponding to the recombination sites of the pXI array expression vector7 to facilitate cloning by homologous recombination) for all selected proteins were codon optimised for expression in E. coli and commercially synthesised and cloned in pUC57 by either Twist Bioscience (sequences < 1.8 kb) or ProteinCT (sequences > 1.8 kb). Synthesised genes were PCR amplified by primers corresponding to the pXI recombination sites and cloned into pXI, in-frame with the vector’s 5’ HA and 3’ HIS tag-encoding sequences, using in vivo recombination. Proteins encoded by each purified plasmid were expressed in vitro (RTS 100 E. coli HY kit – 5 Prime, MD, USA) according to the manufacturer’s instructions and printed onto eight-pad nitrocellulose-coated AVID glass slides (Grace Biolabs, OR, USA) with an Omnigrid 100 microarray printer (Genomic Solutions, Ann Arbor, MI, USA). Vector containing no insert was similarly “expressed” and printed (in multiple locations) to act as a negative control and multiple empty spots were left on each pad to serve as background controls. Purified human IgG, anti-human IgG and parasite extracts (S. haematobium adult- and egg-stage ES products, soluble egg antigen and adult-stage tritonX-100-soluble extract) were also printed as positive controls. Expression quality control was assessed by detection of N-terminal HA and C-terminal HIS tags.