上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 技术与方法 • 上一篇    下一篇

嗜酸乳杆菌ATCC4356 S-层蛋白的克隆、表达和纯化

孙 琴1，曾乃燕2，周爱武2，叶 玮1   

  1. 1.上海交通大学 医学院附属第九人民医院口腔预防儿童科, 上海市口腔医学重点实验室， 上海 200011； 2.上海交通大学 医学院细胞分化与凋亡教育部重点实验室， 上海 200025
  • 出版日期:2014-06-28 发布日期:2014-06-30
  • 通讯作者: 叶 玮, 电子信箱: jyyewei@163.com。
  • 作者简介:孙 琴（1989—）， 女， 硕士生; 电子信箱: sunsqk77@163.com。
  • 基金资助:

    上海第九人民医院院级基金（院2012-04）

The clone, expression, and purification of surface layer protein of Lactobacillus acidophilus ATCC4356

SUN Qin1, ZENG Nai-yan2, ZHOU Ai-wu2, YE Wei1   

  1. 1.Department of Preventive and Pediatric Dentistry, the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011,China; 2.Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Shanghai Key Laboratory of Stomatology, Jiao Tong University School of Medicine, Shanghai 200025, China
  • Online:2014-06-28 Published:2014-06-30
  • Supported by:

    Foundation of the Ninth People's Hospital of Shanghai, 2012-04

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摘要：

目的 克隆嗜酸乳杆菌S层蛋白的编码基因，使其原核表达并纯化表达产物。方法 对嗜酸乳杆菌S层蛋白（S-layer protein）基因进行提取和扩增，连接pET-SUMO3表达载体并转入大肠杆菌内使其表达；采用镍柱亲和层析技术对S层蛋白进行提取及纯化，经SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western blotting验证表达产物。结果 嗜酸乳杆菌ATCC4356的S层蛋白编码基因（1 263 bp）被克隆，且与相关基因有98%同源性。SDS-PAGE及Western blotting证明，重组融合蛋白在细菌中能够高效表达且部分融合蛋白可经亲和层析及酶切去除融合标签获得重组S-层蛋白（43 0000 Da）。结论 通过获得嗜酸乳杆菌S-层蛋白编码基因的方法可实现其原核表达，并得到纯度较高的S层蛋白，为后续的生物学功能研究奠定了基础。

关键词: 嗜酸乳杆菌ATCC4356, S-层蛋白, 克隆, 表达, 纯化

Abstract:

Objective To clone the encoding gene of surface layer (S-layer) protein of Lactobacillus acidophilus, realize the prokaryotic expression, and purify expression products. Methods The gene of S-layer protein of Lactobacillus acidophilus was extracted, amplified, ligated to the expression vector of pET-SUMO3, and transcribed and expressed in E.coli BL21 (DE3). The S-layer protein was extracted and purified by the nickel-affinity chromatography. The expression products were verified by the SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results The encoding gene of S-layer protein of Lactobacillus acidophilus ATCC4356 was cloned and had 98% homology with the relevant gene. Results of the SDS-PAGE and Western blotting showed that the fusion protein expressed efficiently in bacteria. The recombinant S-layer protein (43 000 Da) could be obtained by the affinity chromatography and removing the fusion tag by the enzymatic digestion. Conclusion The prokaryotic expression can be achieved and the S-layer protein with high purity can be acquired by obtaining the encoding gene of S-layer protein of Lactobacillus acidophilus. These are helpful for subsequent studies on biological functions.

Key words: Lactobacillus acidophilus ATCC4356, S-layer protein, clone, expression, purification