Author/Authors :
Thang Long, Phung Faculty of Animal Sciences and Veterinary Medicine - University of Agriculture and Forestry - Hue University, Hue , Vietnam , Viet, Le Quoc Faculty of Animal Sciences and Veterinary Medicine - University of Agriculture and Forestry - Hue University, Hue , Vietnam , Quan, Le Viet Faculty of Animal Sciences and Veterinary Medicine - University of Agriculture and Forestry - Hue University, Hue , Vietnam , Huu Rin, Dong Faculty of Animal Sciences and Veterinary Medicine - University of Agriculture and Forestry - Hue University, Hue , Vietnam , Xuan Hoa, Nguyen Faculty of Animal Sciences and Veterinary Medicine - University of Agriculture and Forestry - Hue University, Hue , Vietnam , Thao, Le Duc Faculty of Animal Sciences and Veterinary Medicine - University of Agriculture and Forestry - Hue University, Hue , Vietnam , Phung, Le Dinh Faculty of Animal Sciences and Veterinary Medicine - University of Agriculture and Forestry - Hue University, Hue , Vietnam , Thi Thu Hien, Nguyen Faculty of Animal Sciences and Veterinary Medicine - University of Agriculture and Forestry - Hue University, Hue , Vietnam , Thi Bich Lan, Dinh Faculty of Animal Sciences and Veterinary Medicine - University of Agriculture and Forestry - Hue University, Hue , Vietnam
Abstract :
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.
Keywords :
Mycoplasma hyopneumoniae , Porcine enzootic pneumonia , P97R1R2 gene fragment , Expression of P97R1R2 protein , Recombinant protein
