Strongyloides stercoralis

Boonroumkaew, Patcharaporn, Sadaow, Lakkhana, Janwan, Penchom, Rodpai, Rutchanee, Sanpool, Oranuch, Buadee, Punyisa, Suprom, Chanida, Thanchomnang, Tongjit, Intapan, Pewpan M. & Maleewong, Wanchai, 2023, Improved diagnostic sensitivity of human strongyloidiasis using point-of-care mixed recombinant antigen-based immunochromatography, Parasite (Paris, France) 30 (60), pp. 1-12 : 3

Strongyloides stercoralis recombinant antigens

The Strongyloides stercoralis L3NieAg.01 ( NIE) sequence (1–621 bp) deposited in the GenBank database (AF136445) was codon-optimized to be suitable for an Escherichia coli expression system and constructed into the pET22.b (+) vector (GenScript, Piscataway, NJ, USA). The recombinant NIE construct was transformed into a cloning host ( E. coli JM 109) (Novagen, Darmstadt, Germany) and an expression host ( E. coli Rosetta-gami 2 (DE3)) (Novagen). The C-terminalfused His-tagged NIE was inoculated into LB broth and induced with 1 mM IPTG and incubated at 37 ° C overnight. Bacterial cells harvested from the culture were centrifuged and then resuspended in a lysis buffer with lysozyme. The suspension was then sonicated on ice. The recombinant NIE protein was initially in insoluble form and was dissolved in 8 M urea, pH = 8 (Elago Enterprises Pty Ltd., Cherrybrook, NSW, Australia).

The solubilized NIE protein fused with 6 × His-tags at the C-terminal was purified by affinity chromatography using HisTrap™ HP 1 mL columns in the ÄKTA start protein purifi- cation system as recommended by the manufacturer (Global Life Sciences Solutions USA LLC, Marlborough, MA, USA). The purified NIE protein was examined on a 12% SDS– PAGE, and the protein concentration was measured using the Bradford Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin as a standard.

The Strongyloides recombinant IgG-immunoreactive (SsIR) antigen was prepared as reported previously [ 4]. The purified SsIR protein was examined on a 10% SDS– PAGE. Each recombinant antigen was stored at 4 ° C before use.