The 24th International Pig Veterinary Society Congress

Secretory expression of 46-kilodalton surface antigen from Mycoplasma hyopneumoniae in Bacillus subtilis

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

[Introduction] Mycoplasma hyopneumoniae is the causative agent of swine enzootic pneumonia (SEP), one of the most important chronic respiratory diseases that affects swine production worldwide. Vaccination is the most effective method for control of SEP. A plethora of studies indicated that 46 kDa surface antigen (P46), a highly conserved and immunodominant antigen, is considered to be a potential vaccine candidate. Our previous study has cloned and expressed recombinant P46 (rP46) in Escherichia coli. However, the purification of rP46 from E. coli is time-consuming. In comparison to E. coli, Bacillus subtilis is a more attractive host because it has the capacity to secrete proteins into the growth medium, which significantly simplify the downstream processing. Therefore, the aim of this study is to use B. subtilis as a host for secretory expression and one-step purification of rP46. [Materials and Methods] Silent mutated P46 gene which the nonsense TGA codons in the gene had been converted to TGG codons (tryptophan) was amplified by PCR from pET-P46 and cloned into five different secretion vectors. The resulting plasmids were transformed into B. subtilis WB800N/pBL1 using electroporation, respectively. Transformants were incubated with shaking at 30°C to OD600=0.4-0.5 followed by induction with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 48 hours at 30°C. Supernatant samples of B. subtilis transformants were collected and analyzed by SDS-PAGE and western blot. Recombinant P46 (rP46) produced by B. subtilis (pSP2-P46) was purified from culture supernatant by immobilized metal-ion affinity chromatography (IMAC). Purity of recombinant P46 was determined by SDS-PAGE. [Results and Discussion] A 1.2-kb DNA fragment encoding mature P46 with a C-terminal His-tag was amplified by PCR and cloned into five secretion plasmids. The resulting plasmids, pSP1-P46 to pSP5-P46, were transformed into B. subtilis and used for expression of the rP46 protein. The expression of the rP46 by B. subtilis transformants were achieved by addition of IPTG into the culture medium. Forty-eight hours after induction with IPTG, secretory expression of rP46 were observed for all transformants. The secretory expression of rP46 reached the highest level when P46 fused to the SP2 signal peptide. The rP46 could be directly purified from the supernatant of B. subtilis (pSP2-P46) by using IMAC with a yield of 22.55 mg/L and a purity of >95%. [Conclusion] This study presents the high-level secretory expression and one-step purification of rP46 in B. subtilis. The purified rP46 will be further used in subunit vaccine and enzyme-linked immunosorbent assay (ELISA) kit developments.