The 24th International Pig Veterinary Society Congress

Extracellular production of xylose-binding lipoprotein from Mycoplasma hyopneumoniae in Bacillus subtilis

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

[Introduction] Mycoplasma hyopneumoniae is the primary etiological agent of swine enzootic pneumonia (SEP), one of the most prevalent respiratory diseases in pigs worldwide. Vaccination is considered as an important and effective tool to control SEP. Commercially available vaccines against M. hyopneumoniae infection comprise inactivated whole-cell bacterins. However, production of these vaccines employing the complicated cultivation method are expensive and time consuming. The use of specific antigens as subunit vaccines produced by recombinant DNA technology may be a preferable alternative to the use of bacterins. Our previous study showed that xylose-binding protein (XylF) is a potential subunit vaccine candidate. The gene encoding XylF was cloned and expressed in Escherichia coli. However, the purification of recombinant XylF (rXylF) from E. coli is time-consuming. In the present study, we demonstrated that Bacillus subtilis is an attractive alternative for high-level expression and simplified purification of rXylF. [Materials and Methods] Silent mutated XylF gene which the nonsense TGA codons in the gene had been converted to TGG codons (tryptophan) was amplified by PCR from pET-XylF and cloned into three different secretion plasmids. The resulting plasmids, pSP1-XylF to pSP3-XylF, were transformed into B. subtilis WB800N/pBL1, respectively. Transformants were incubated with shaking at 30°C to an OD600 of 0.4-0.5, followed by induction 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 rXylF produced by B. subtilis (pSP3-XylF) was purified from culture supernatant by immobilized metal-ion affinity chromatography (IMAC). Purity of rXylF was determined by SDS-PAGE. [Results and Discussion] A 1.3-kb DNA fragment encoding mature XylF with a C-terminal His-tag was cloned into three secretion plasmids. The resulting plasmids were transformed into B. subtilis, respectively. The expression of the rXylF by B. subtilis transformants were achieved by addition of IPTG. After 48 hrs of IPTG induction, a significant amount of rXylF accumulated in the supernatants of all transformants were observed. The highest secretory expression level of rXylF was achieved when the XylF fused to the SP3 signal peptide. The rXylF was purified from the supernatant of B. subtilis by using IMAC with a yield of 19.81 mg/L and a purity of >95%. [Conclusion] This study presents the extracellular production and purification of rXylF in B. subtilis. The purified rXylF will be further used in development of a subunit vaccine or cocktail vaccine.