Cloning and characterization of HcGOB gene from Haemonchus contortus

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.