Cloning and Optimizing the Culture Parameters for Expression of R1 and R2 Repeat Regions of P97 Adhesin from Mycoplasma hyopneumoniae in Escherichia coli

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the causative agent of porcine enzootic pneumonia (PEP), a chronic respiratory disease that affects pigs of all ages worldwide and causes considerable economic losses in the pig industry. The R1 and R2 repeat regions of P97 adhesin (P97R1R2) of M. hyopneumoniae play important roles in adherences to the host cells to initiate the infection process, and are capable to confer immunogenicity, and potential candidates for development of the recombinant subunit vaccines. The objective of this study was to clone P97R1R2 gene fragment of M. hyopneumoniae isolated from pigs in Thua Thien Hue province, Vietnam and optimize culture parameters (cultivation temperature, culture media, inducer concentration, induction time and incubation time) for improving expression of recombinant P97R1R2 protein in E. coli. The result showed that the nucleotide sequence of P97R1R2 gene fragment of M. hyopneumoniae was 779 bp in length, corresponding to 272 amino acids with 10 repeats of AAKPE(V) in R1 and 3 repeats of GS(A) PN (S) QGKKAE in R2. Expression of the P97R1R2 in E. coli BL21 StarTM (DE3) produced a fusion protein with molecular weights of approximately 33.42 kDa (including 3.7 kDa of fusion fragment of pET-200/D-TOPO vector). Furthermore, the cultivation temperature at 25o C, YJ medium, Isopropulβ-D-1-thiogalactopyranoside (IPTG) concentration of 0.6 mM, induction time at an optical density of 1.5 at 600 nm, and post-induction incubation time for 6 h were determined to be the optimal for the expression of target protein. In conclusion, this study was successful in cloning P97R1R2 gene fragment of M. hyopneumoniae isolated from pigs in Thua Thien Hue province, Vietnam and optimizing selected culture parameters for higher expression of recombinant P97R1R2 protein in E. coli BL21 StarTM (DE3) cells.