Table I.
Bacterial strains, primers, and plasmids used in this study, in which the tonB2 gene was cloned from Actinobacillus pleuropneumoniae JL01 (serovar 1) and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21(DE3)
| Strain, plasmid, and primer | Relevant characteristics | Source |
|---|---|---|
| A. pleuropneumoniae | ||
| JL01 | Serovar 1 | Isolated from Jilin province, China |
| 4074 | Serovar 1 | Pat Blackall |
| SLW03 | Serovar 1 | Lin et al (12) |
| E. coli | ||
| DH5a | Cloning vehicle: supE44 □lacU169 (ϕ80 lacZ□M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 | Takara (Dalian, China) |
| BL21(DE3) | Expression host: F− ompT r−B m−B; DE3 is a λ derivative carrying lacI and T7 RNA polymerase genes under placUV5 control | Takara |
| Primers | ||
| P1 | 5′ GGG GGA TCC ATG AAG AAA AAA CAT TCT CG 3′: upstream primer with BamHI site (underlined) comprising positions 1 to 20 of tonB2 coding sequence | This work |
| P2 | 5′ GGG GTC GAC TTA TTC AAT CGA GAA TTT CAC C 3′: downstream primer with SalI site (underlined) comprising positions 837 to 858 of tonB2 coding sequence | |
| P3 | 5′ GGC TGT CTG TTA GTG GTT CG 3′: upstream primer comprising positions 953 to 972 of apxIVA coding sequence | Liu et al (13) |
| P4 | 5′ CCG TGT GCA GAA ATA CTG CC 3′: downstream primer comprising positions 2125 to 2144 of apxIVA coding sequence | |
| Plasmids | ||
| pMD18-T | E. coli cloning vector carrying an ampicillin resistance determinant | Takara |
| pMD-tonB2 | pMD18-T carrying the tonB2 gene open reading frame (ORF) of A. pleuropneumoniae JL01, for sequence analysis | This work |
| pGEX-KG | N-terminal glutathione-S-transferase (GST) fusion expression vector: pBR322 ori, Ampr | Guan et al (14) |
| pKG-tonB2 | pGEX-KG carrying the tonB2 gene ORF of A. pleuropneumoniae JL01, for GST-TonB2 expression | This work |