TABLE 2.
PCR primers used to construct STa recombinant, STa mutant, and pLT192:pSTa12 and pLT192:pSTa12 chimeric strainsa
| Primer | Sequence (5′ to 3′) | Description |
|---|---|---|
| 184EcoRV-F | GTCAGGCACCGTGTATGAAAT | Plasmid pACYC184 sequence at the EcoRV site |
| pBREagI-R | GTCCCTGATGGTCGTCATCT | Plasmid pACYC 184 sequence downstream at EagI site |
| STaSfcI-F | GTGAAACAACCTGTAGGGA | To amplify native estA gene, cloned into pACYC184 |
| STaEagI-R | GTGGAGCCGGCCGAAACA | To amplify native estA gene, cloned into pACYC184 |
| STaHindIII-F | TGCAAAATAAGCTTAACTAATCTC | To amplify native estA gene, cloned into pUC19 |
| STaBamH1-R | GTGGAGCCGGATCCAACAG | To amplify native estA gene, cloned into pUC19 |
| pSTa11K-F | GAACTTTGTTGTAAACCTGCCTGT | Pairs with pBREagI-R to mutate the 11th amino acid |
| pSTa11K-R | ACAGGCAGGTTTACAACAAAGTTC | Pairs with 184EcoRV-F to mutate the 11th amino acid |
| pSTa12F-F | CTTTGTTGTAATTTTGCCTGTGCC | Pairs with pBREagI-R to mutate the 12th amino acid |
| pSTa12F-R | GGCACAGGCAAAATTACAACAAAG | Pairs with 184EcoRV-F to mutate the 12th amino acid |
| pSTa13Q-F | TGTTGTAATCCTCAGTGTGCTGGA | Pairs with pBREagI-R to mutate the 13th amino acid |
| pSTa13Q-R | TCCAGCACACTGAGGATTACAACA | Pairs with 184EcoRV-F to mutate the 13th amino acid |
| pLT:STa-F | GCAATCAGTGGGCCGGGGCCCATGAACAACTTTTACTGCTG | 3′ end of LT + linker + 5′ end of STa |
| pLT:STa-R | GTTGTTCATGGGCCCCGGCCCACTGATTGCCGCAATTGAATTGG | 5′ end of STa + linker + 3′ end of LT |
| STa12NS-R | ATAACATCCAGCACAGGCAAAATT | STa12 mutant without stop codon, for TA cloning |
| ST13NS-R | ATAACATCCAGCACACTGAGG | STa13 mutant without stop codon, for TA cloning |
| LT-R | GTTTTTCATACTGATTGCCCCAATTG | To amplify LTAB genes without stop codon |
| STa-F | AACAACACATTTTACTGCTGTG | To amplify the STa gene without signal peptide |
| hLT-F | ATGATTGACATCATGTTGCATATAGG | To amplify LT192:mSTa fusion genes, for TA cloning |
a
Bold indicates the target amino acid to be mutated for STa toxoids, underlining indicates the enzyme restriction site, and italic indicates the Gly-Pro-Gly-Pro linker. Primers pLT:STa-F and pLT:STa-R were used to fuse the mutated porcine eltAB (encoding pLT192 protein) with the mutated porcine estA (encoding STa12 or STa13 protein) genes.