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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bsh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。
Objective To clone and overexpress the gene of bile salt hydrolase to study the effect of bile salt hydrolase on the bile salt tolerance and the rate of cholesterol degradation. Methods The bile salt hydrolase gene was overexpressed in Escherichia coli BL21 (DE3) and Bifidobacterium longum NCC2705 by routine prokaryotic expression method, and the bile salt tolerance and hypocholesterolemic activity of Bifidobacterium longum NCC2705 and pMgap-bsh / NCC2705 experiment. Results The bile salt hydrolase gene of NCC2705 was successfully expressed in E. coli. The SDS-PAGE of purified GST tag and GST fusion protein showed that the relative molecular mass of bile salt hydrolase was about 35 × 103. Strain pMgap-bsh / NCC2705 overexpressing bsh showed slightly higher bile salt tolerance and cholesterol degradation rate than the standard strain NCC2705. Conclusion The constructed bile salt hydrolase-producing strain may be beneficial to improve the bile salt tolerance and cholesterol-lowering ability of the bacteria. The cholesterol-lowering pathway of probiotics is not single, and the degradation rate of cholesterol Greater impact.