Dh5α strain of escherichia coli

The DH5α strain of Escherichia coli (Invitrogen) was used for cloning and amplifying plasmid DNA. The strains of crystalliferous B. thuringiensis subsp. israelensis (Bti) 4Q5, acrystalliferous Bti 4Q7, and L. sphaericus (Ls) 2363 were obtained from the Bacillus Genetic Stock Center (Ohio State University, Columbus, OH). Erythromycin-resistant recombinants 4Q7/pWF45 and 4Q7/p45S1, producing, respectively, Cyt1Aa and BinA/BinB (42 kDa/51 kDa) parasporal bodies have been described previously^{13 (link), 30 (link)–32 (link)}. All strains were maintained on Nutrient agar (Becton Dickinson, Sparks, MD) throughout the study. LB medium (Becton Dickinson, Sparks, MD) was used for growing E. coli and extracting plasmid DNA using the Wizard Plus Mini-prep DNA Purification system (Promega). Genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen).

In silico analysis of NSs nucleolar localization (NoLS) motif was performed while using the NoD software, an online-available algorithm (http://www.compbio.dundee.ac.uk/www-nod/) developed by G.J. Barton [28 (link)]. The algorithm of trained artificial neural network allowed for the prediction of NoLS regions, characterized by a predetermined threshold of 0.8. In parallel, the prediction of intrinsically disordered region of NSs proteins was obtained by amino acid sequence analysis with the PONDR^® online software (http://www.pondr.com/).
Site directed-mutagenesis was performed on 50 ng of tagged-NSs plasmids, while using PFU Ultra HF polymerase (Agilent, Santa Clara, CA, USA), in order to validate predicted nucleolar addressing sequence identified in NSs protein. Table S1b lists the primers designed on Agilent website (https://www.agilent.cm/store/primerDesignProgram.jsp). The reactions were carried out in a thermal cycler, as follows: denaturation at 95 °C for 30 s followed by 18 cycles each for 30 s at 95 °C, 1 min at 55 °C, and 5 min at 68 °C. To get rid of the plasmid used as DNA matrix, the amplified products were treated with DpnI restriction enzyme for 2 h at 37 °C. Plasmids were then amplified in DH5α strain of Escherichia coli (Invitrogen).

The DNA vaccine construct was modified from pQE31/Sjc26GST, which contains the intact open reading frame (ORF) of 26 kDa SjGST from Schistosoma japonicum (GenBank accession no. BU711548.1) [13 (link)]. Briefly, the intact ORF of Sjc26GST was amplified by PCR using the pQE31/Sjc26GST vector as a template and primers containing EcoRV and BamHI restriction sites (Forward-5′-CGGGATCCCGTCATGTCCCCTATACTAGGTTAT-3′ and Reverse-5′-GGGATATCCCTTTATTTTGGAGGATGGTCGCCA-3′). The gene products was digested with BamHI/EcoRV at 37°C for 4 h, and then subcloned into the pcDNA4 vector (Invitrogen, Waltham, MA, USA) at 37°C overnight to generate the pcDNA/SjGST construct. Constructs were confirmed by restriction analysis and sequencing (Fig 1A). The DH5α strain of Escherichia coli (Invitrogen, USA) was transformed with plasmid DNA. The DNA vaccine was amplified and purified according to the manufacturer’s instruction for endotoxin-free Giga Prep Kits (Qiagen, Valencia, CA, USA). The plasmid DNA vaccine was resuspended in 5% glucose and stored at −80°C until use. In addition to pcDNA/SjGST, we used rSjGST, prepared previously (Fig 1B) [13 (link)], and an IL-12-encoding plasmid (pIL-12), kindly provided by the laboratory of Dr. Tauo [32 (link)], as an adjuvant to enhance the immune response.