长双歧杆菌NCC2705中胆盐水解酶基因的克隆表达及其特性的研究

摘要

Objective To clone and overexpress bile salt hydrolase of Bifidobacterium longum NCC2705 in E. coli BL2l( DE3 ) and to test the bile salt tolerance and cholesterol-lowering efficiency of B. longum NCC2705 and pMgap bile salt hydrolase gene ( bsh )/NCC2705. Methods A conventional prokaryotic expression method was used to clone and express bile salt hydrolase gene in E. coli BL21 ( DE3 ) and B. longum NCC2705. Bile salt tolerance and cholesterol-lowering ability of B. longum NCC2705 and pMgap-bs/i/NCC2705 were explored. Results The bile salt hydrolase gene was suc-cessfully expressed,and GST-bsh fusion protein was well purified. The relative molecular mass of bile salt hydrolase was about 35 X 103 according to the results of SDS-PAGE gel electrophoresis of purified GST-bsh fusion protein and GST empty tag of expression broth supernatant of pGEX-4T-l-6sh/BL21( DE3 ). Bile salt tolerance and cholesterol-lowering ability of pMgap-6sh/NCC2705 was higher than that of the standard strain NCC2705. Conclusion The strains of overexpressed bile salt hydrolase may help to improve the bile tolerance and cholesterol-lowering ability of strains. Probioticis does not lower cholesterol in a single way,which is related to the bile salt concentration of the medium.%目的 克隆并过表达胆盐水解酶基因,研究胆盐水解酶对菌体胆盐耐受性和胆固醇降解率的影响等特性.方法 采用常规原核表达方法在大肠杆菌BL21(DE3)和长双歧杆菌NCC2705中过表达胆盐水解酶基因,并进行长双歧杆菌NCC2705和pMgap-bsh/NCC2705的胆盐耐受性和降胆固醇实验.结果 在大肠杆菌中成功表达了NCC2705的胆盐水解酶基因,经纯化的GST空标签和GST融合蛋白的SDS-PAGE电泳结果显示,胆盐水解酶的相对分子质量约为35×103;构建的过表达bah的菌株pMgap-bsh/NCC2705比标准菌株NCC2705表现出了略高的胆盐耐受性和胆固醇降解率.结论 所构建的高效表达胆盐水解酶菌株可能有利于提高菌体的胆盐耐受性和降胆固醇能力,益生菌的降胆固醇途径不是单一的,培养基中是否含有胆盐对胆固醇的降解率影响较大.