Plasmid construction.

The 1,023-bp coding sequence (CDS) of HcGAPDH was amplified from the total cDNA of H. contortus by PCR using primers previously described (10). PCR products were sequenced in both directions (BioSune, China) after being cloned to the pET-32a vector (TaKaRa, China), resulting in the pET32a-HcGAPDH plasmid.

To generate a recombinant spore carrying the CotB-HcGAPDH, genomic DNA of B. subtilis strain 168 was used as the template to amplify the CotB gene of 1,088 bp, which included the promoter of 263 bp and a partial N-terminal CDS of 825 bp. The PCR primers are listed in Table S1. PCR products were cloned into the pMD 18-T vector (TaKaRa, China). The CotB and HcGAPDH genes were fused in order and cloned into the pDG364 vector (pDG364-CotB-HcGAPDH). The CotB-HcGAPDH was amplified by PCR with primers (Table S1). The fused CotB-HcGAPDH was subcloned into E. coli-B. subtilis shuttle vector pDG364 (Miaolingbio, China), resulting in pDG364-CotB-HcGAPDH plasmid. The control plasmid pDG364-CotB was constructed by cloning the CotB gene to the pDG364 vector. All cloned DNAs were confirmed without mutations by sequencing (BioSune, China).

Primers used in the present study. Download Table S1, DOCX file, 0.01 MB.