Table 1 Strains, plasmids, and primers used in this study
Strain, plasmid and primer | Relevant characteristics | Source |
|---|---|---|
A. pleuropneumoniae | ||
SLW01 | Serovar 1 | Weicheng Bei |
13039 | Serovar 10 | Pat Blackall |
E. coli | ||
DH5a | Cloning vehicle: supE44 ΔlacU169 (φ80 lacZΔM15) hsdR17recA1 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 (Dalian, China) |
Plasmid | ||
pMD18-T | E. coli cloning vector carrying an ampicillin resistance determinant | Takara (Dalian, China) |
pMD-lip40 | pMD18-T carrying lip40 gene of A. pleuropneumoniae strain SLW01. | This work |
pGEX-KG | N-terminal glutathione S-transferase (GST) fusion expression vector: pBR322 ori, Ampr | [19] |
pGEX-lip40 | pGEX-KG carrying the lip40 gene for over-expression Lip40 protein | This work |
Primers (Primers were all synthesized in Sangon, Shanghai, China) | ||
P1 | 5′ ATG AAA AAC ATC ACA AAA TTT GCA G 3′, upstream primer for lip40 gene cloning | This work |
P2 | 5′ TTA CTT TTG TTG TTT TGC GCC AAA 3′, downstream primer for lip40 gene cloning | This work |
P3 | 5′ CGG TTC GAT TTG GTG TGT ATG A 3′,upstream primer of lip40 gene for qRT-PCR analysis | This work |
P4 | 5′ AAC AAG TAA GCA TCA CCT GTG T 3′, downstream primer of lip40 gene for qRT-PCR analysis | This work |
P5 | 5′ AAG TGG CAG AGC TGG AAG AT 3′, upstream primer of internal control gene rluC for qRT-PCR analysis | This work |
P6 | 5′ TCA CAC CAA AAC TCA AGC CG 3′, downstream primer of internal control gene rluC for qRT-PCR analysis | This work |
P7 | 5′ TTG GAT CCT GTG GCA GTA AGA ACC ATT C 3′, upstream primer with BamHI site (underlined) comprising positions 58 to 77 of lip40 coding sequence | This work |
P8 | 5′ GGA AGC TTT TAC TTT TGT TGT TTT GCG C 3′, downstream primer with HindIII site (underlined) comprising positions 878 to 897 of lip40 coding sequence | This work |