分别将枯草芽孢杆菌(Bacillus subtilis 168)芽孢衣壳蛋白CotB、CotC、CotG和CotX的启动子和编码序列与来自嗜热脂肪芽孢杆菌(Bacillus stearothermophilus IAM11001)的β-半乳糖苷酶基因bgaB进行重组,构建融合表达cotB-bgaB、cotC-bgaB、cotG-bgaB和cotX-bgaB的整合型重组质粒。将4种重组质粒分别转入枯草芽孢杆菌Bacillus subtilis 168(trp-),获得了能在芽孢表面展示的重组菌株PB701、PB702、PB703和PB704。经Western blot检测,4种重组菌株均表达了预期分子量的融合蛋白,初步表明β-半乳糖苷酶被锚定在重组菌株的芽孢表面。以oNPG为底物测定4种重组菌株芽孢表面展示β-半乳糖苷酶的水解能力,得到的酶活分别为0.14、0.06、0.22和0.20 U/mL。

In this work,we developed an efficient spore display system that a model protein β-galactosidase was anchored on the spore surface of Bacillus subtilis 168 based on the use of spore coat proteins.The PCR-amplifying cotB,cotC,cotG and cotX were ligated with pMD-19T and digested with XbaI and KpnI,and then subcloned into vector pJS700a previously digested with the same two restriction enzymes,finally resulted in the plasmids pJSB,pJSC,pJSG and pJSX.To construct the gene fusions,the bgaB from Bacillus stearothermophilus IAM11001 was cloned into the KpnI and EcoRI sites of plasmid pJSB,pJSC,pJS G and pJSX to generate generating the plasmids pJSBB,pJSCB,pJSGB and pJSXB,respectively After linearization with BglII restriction endonuclease,the four recombinant integrative plasmids were transformed into B.subtilis 168 to yield the recombinant strain PB701,PB702,PB703 and PB704,respectively.Results from Western blot analysis showed that the fusion protein was immobilized on the spore surface.Using oNPG as substrate,the enzyme activity of spore-displaying β-galactosidase was assayed and they were 0.14,0.06,0.22 and 0.20 U/mL for PB701,PB702,PB703 and PB704,respectively.This result suggested that the fusion proteins could be successfully expressed and located on the spore surface of B.subtilis.