胆盐水解酶基因的克隆及其在干酪乳杆菌CECT5276中的分泌表达

本试验旨在克隆植物乳杆菌(L.plantarum)DPP8的胆盐水解酶(BSH)基因,并在干酪乳杆菌(L.casei)CECT5276中对其进行表达.通过PCR技术克隆L.plantarum DPP8的BSH基因,然后将该基因重组到L.casei表达载体pMJ67-sp中,获得了重组表达质粒pMJ67-sp-BSH,再...

Cloning of Bile Salt Hydrolase Gene and Its Secreted Expression in Lactobacillus casei CECT5276

The purpose of this experiment was to clone the bile salt hydrolase(BSH)gene from Lactobacillus plantarum(L.plantarum)DPP8 and express it in Lactobacillus casei(L.casei)CECT5276.The BSH gene from L.plantarum DPP8 was cloned by PCR technique,and then recombined it into L.casei expression vector pMJ67-sp and yielding recombinant expression vector pMJ67-sp-BSH.The above recombinant expression vector was elect-transferred into L.casei CECT 5257 and inducible secretion expressed with lactose.The BSH activity was detected by taurine standard curve method,and the cholesterol degradation ability was evaluated using o-phthalaldehyde(OPA)method.The results showed that the full-length sequence of BSH gene cloned in this experiment was 975 bp.It shared 99.6%sequence homology with BSH gene from L.plantarum MBUL69 by homology comparison,and lied in the same branch with L.plantarum MBUL69 BSH gene by phylogenetic tree analysis.The sodiumdodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)result showed that the molecular weight of recombinant protein was approximately 37 ku,which was consistent with the expected size.The BSH activity was 42.57 U/mL in recombinant L.casei CECT5276(pMJ67-sp-BSH)under the conditions of 37℃,0.5%lactose and inducted 36 h.The degradation rate of cholesterol was 94.43%when recombinant L.casei CECT5276(pMJ67-sp-BSH)cultured for 48 h in the medium containing 2%cholesterol.In conclusion,the BSH gene from L.plantarum DPP8 is cloned successfully,and the high efficiency expression and secretion of BSH in L.casei CECT5276 is achieved in this experiment.