The 24th International Pig Veterinary Society Congress

Secretory production of sugar ABC transporter substrate-binding protein from Mycoplasma hyopneumoniae in Bacillus subtilis

JP Wang, MW Hsieh, JF Lai, ZW Chen, WZ Huang, HJ Lin, JH Lin

[Introduction] Mycoplasma hyopneumoniae is the principal aetiological agent of swine enzootic pneumonia, a chronic respiratory disease occurs worldwide. Control of M. hyopneumoniae infections can be accomplished by vaccination. Currently available M. hyopneumoniae vaccines are made from whole inactivated M. hyopneumoniae bacteria. However, production of these vaccines are complicated, expensive and time-consuming. Recently, considerable effort is being made to develop low cost easily-produced subunit vaccines. Our previous study showed that sugar ABC transporter substrate-binding protein (Mhp145) from M. hyopneumoniae is considered to be an attractive vaccine candidate. The gene encoding Mhp145 has been cloned and expressed in Escherichia coli. However, the purification of recombinant Mhp145 (rMhp145) from E. coli is time-consuming and labor-intensive. The objective of this study is to extracellularly express rMhp145 in Bacillus subtilis and establish a simple purification procedure for rMhp145. [Materials and Methods] Silent mutated Mhp145 gene which the nonsense TGA codons in the gene had been converted to TGG codons (tryptophan) was amplified by polymerase chain reaction from pET-Mhp145 and cloned into four secretion plasmids. The resulting plasmids, pSP1-Mhp145 to pSP4-Mhp145, were transformed into B. subtilis WB800N/pBL1, respectively. Transformants were incubated with shaking at 30°C to an OD600 of 0.4-0.5 and then induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 48 hours at 30°C. The supernatant samples of B. subtilis transformants were collected and analyzed by SDS-PAGE and western blot. The rMhp145 produced by the B. subtilis (pSP2-Mhp145) was purified from culture supernatant by immobilized metal-ion affinity chromatography (IMAC). Purity of rMhp145 was determined by SDS-PAGE. [Results and Discussion] A 1.2-kb DNA fragment encoding mature Mhp145 with a C-terminal His-tag was cloned into four secretion plasmids. The resulting plasmids were transformed into B. subtilis, respectively. The expression of the rMhp145 by B. subtilis transformants was achieved by IPTG addition. Forty-eight hours after induction, a significant amount of rMhp145 were noticed in the supernatants of all transformants. The highest secretory expression level of rMhp145 was achieved when the Mhp145 fused to the SP2 signal peptide. The rMhp145 could be directly purified from the supernatant of B. subtilis by using IMAC. The final yield of rMHP145 was 18.31 mg/L with purity above 95%. [Conclusion] This study demonstrated the secretory production and purification of rMhp145 in B. subtilis. The purified rMhp145 will be further used in development of a subunit vaccine or cocktail vaccine.