Gel extraction kit

Adult H. contortus worms were obtained as previously described [33 ]. Total RNA was extracted from adult worms by the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) method [34 ]. The complementary DNA (cDNA) was synthesized by HiScript III Kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Based on the sequences of H. contortus (GenBank: HF967182.1), the HcGOB gene was amplified by specific primers (GOB-F and GOB-R, Additional file 1: Table S1). PCR assays were performed in a total reaction volume of 50 μl [25 μl 2× primer master mix (Takara, Dalian, China), 19 μl ddH2O, 2 μl cDNA, 2 μl of each primer) and amplification was conducted according to the kit instructions, as described by Aleem et al. [35 (link)]. The gene product was purified by using the Gel Extraction Kit (Vazyme, Nanjing, China) and ligated into the expression plasmid pET28a(+) vector (Novagen, Madison, WI, USA). The recombinant plasmid, pET28a (+)/HcGOB, was identified through digestion by restriction enzymes (BamHI and HindIII), and the sequence was analyzed with online BLAST analysis.

Restriction and other enzymes were purchased from Takara Bio (Shiga, Japan). The one-step cloning kit for ligation and the gel extraction kit were purchased from Vazyme Biotech Inc. (Nanjing, China), and the plasmid mini kit was purchased from Omega Bio-Tek (Norcross, GA, USA). All other chemicals were commercially available.

To amplify the Class 1 integron, forward (5′-ATCATCGTCGTAGAGACGTCGG-3′) and reverse primers (5′-GTCAAGGTTCTGGACCAGTTGC-3′) were used as described previously by Rosser et al. [36 (link)]. The primer pair for 16SrRNA (forward 5’-CGGTGAATACGTTCYCGG-3’ and reverse primer 5’-GGTACCTTGTTACGACTT-3) was used as described by Gaze et al. [37 (link)]. The PCR reactions were conducted in a reaction volume of 50 µL, and the amplified fragments were separated using gel electrophoresis for visualization. The different bands were cut from the gel and extracted using a gel extraction kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The DNA fragments were cloned into a blunt cloning vector using a TA/Blunt-Zero Cloning Kit (Vazyme, Nanjing, China), according to the manufacturer’s instructions, and accordingly transferred into E. coli DH_5α competent cells. These cells were then cultured overnight on LA medium containing ampicillin at a concentration of 100 µg/mL. The positive colonies were selected after overnight incubation, and the plasmids were extracted from the culture using a plasmid extraction kit (Vazyme, Nanjing, China). These recombinant plasmids were Sanger sequenced via a universal sequencing primer at AuGct Biotechnology Co. Ltd. (Beijing, China).