重组ETEC k88ac-LTB干酪乳杆菌在人工消化液中的生存性能

Survival properties of ETEC surface-displayed K88ac-LTB on Lactobacillus casei

摘要:

探讨重组干酪乳杆菌(Lactobacillus casei)在人工消化液中的存活能力。将K88ac-LTB基因从表达载体pQE-30克隆到L. casei细胞表面表达载体pLA中, 构建了重组表达载体pLA-K88ac-LTB, 并将其电转化至L. casei中, 在MRS培养基中培养后, Western blotting检测, 有约71.2 kD蛋白得到了表达, 表达蛋白的大小与理论值相符且可被抗血清所识别, 间接免疫荧光实验及流式细胞结果表明, 多聚谷氨酸合成酶A蛋白(pgsA)能够将融合蛋白成功地展示在菌体表面。将重组菌接种于人工模拟的胃肠液中, 通过平板计数观察其存活能力。结果表明, 重组干酪乳杆菌在人工消化液中均具有良好的存活性能, 符合益生菌的基本特征, 为实验动物模型的建立奠定了基础。

Abstract:

K88ac-LTB gene derived from pQE30-K88ac-LTB was cloned into the expression vector pLA and then the recombinant vector was transformed into the competent cells Lactobacillus casei 525. The recombinant bacteria were grown at 37°C, in MRS broth. Western blotting analysis with rabbit-anti-K88ac-LTB polyclonal serum indicated that the recombinant protein reacted with the specific antibodies. The results showed that the molecular weight of the recombinant protein was about 71.2 kD. The K88ac-LTB fusion protein on the cell surface was confirmed by immunofluorescence mciroscopy and flow cytometric analysis. In addition, the survival of recombinant Lactobacillus casei 525 was studied in imitative gastrointestinal environments such as artificial gastro fluid (pH 1.5–5.5), artificial intestinal fluid, bile(0.3–3.0 g/L).The results indicated that the recombinant strain survived well in artificial gastric fluids at pH 2.5–4.5 in 5 h. The recombinant Lactobacillus casei 525 could slowly grow in the artificial intestinal fluid for different time, and could survive in 0.3%bile.