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

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 Star TM (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 25 o 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 Star TM (DE3) cells.Citation | Long PT, Viet LQ, Quan LV, Rin DH, Hoa NX, Thao LD, Phung LD, Hien NTT and Lan DTB (2019). Cloning and optimizing the culture parameters for expression of R1 and R2 repeat regions of P97 adhesin from Mycoplasma hyopneumoniae in Escherichia coli.

Cloning of the Gene Encoding Mycoplasma hyopneumoniae P36 Protein, Expression and Evaluating the Antigenicity of Recombinant Protein

“…The gene encoding M. hyopneumoniae P36 protein from the 1% agarose gel was purified using TopPURE® PCR/ GEL DNA PURIFICATION KIT (ABT, Vietnam) following the manufacture's instruction. Techniques for insertion of the target gene into pGEM ® -T Easy vector, transformation of the recombinant plasmid into competent E. coli TOP 10 cells, selection and confirmation of positive colonies, biomass production, and extraction of recombinant pGEM ® -T Easy/P36 vector were carried out as described by Phung et al (2019). The gene encoding M. hyopneumoniae P36 protein was sequenced by Genlab Company (Vietnam).…”