分子与细胞营养 MOLECULAR AND CELLULAR NUTRITION

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

  • 丁轲 ,
  • 余祖玲 ,
  • 王镨蒂 ,
  • 余祖华 ,
  • 李旺 ,
  • 李元晓 ,
  • 何万领 ,
  • 曹平华 ,
  • 张春杰 ,
  • 刘宁
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  • 1. 河南科技大学宏翔生物饲料实验室, 洛阳 471003;
    2. 洛阳市活载体生物材料与动物疫病防控重点实验室, 洛阳 471003
丁轲(1977-),男,河南永城人,副教授,博士,主要从事动物微生态与动物传染病学研究。E-mail:keding19@163.com

收稿日期: 2020-06-28

  网络出版日期: 2021-01-18

基金资助

国家自然科学基金项目(32072771);河南省自然科学基金项目(182300410052)

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

  • DING Ke ,
  • YU Zuling ,
  • WANG Pudi ,
  • YU Zuhua ,
  • LI Wang ,
  • LI Yuanxiao ,
  • HE Wanling ,
  • CAO Pinghua ,
  • ZHANG Chunjie ,
  • LIU Ning
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  • 1. Hongxiang Biological Feed Laboratory, Henan University of Science and Technology, Luoyang 471003, China;
    2. Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control, Luoyang 471003, China

Received date: 2020-06-28

  Online published: 2021-01-18

摘要

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,再将其电转化至L.casei CECT5276中,通过乳糖诱导BSH分泌表达。采用牛磺酸标准曲线法对表达产物的BSH活力进行测定,并采用邻苯二甲醛(OPA)法对其降胆固醇能力进行测定。结果显示:本试验获得了BSH基因的全长序列,为975 bp;同源性比较显示该基因与L.plantarum MBUL69的BSH基因的同源性为99.6%;进化树分析显示该基因与L.plantarum MBUL69的BSH基因处于同一分支。表达产物的十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示获得了1条分子质量约为37 ku的蛋白条带,与推测的BSH蛋白分子质量大小相符。酶活力测定结果表明重组菌株在37℃、0.5%乳糖诱导36 h时,表达产物中BSH活力最高,可达42.57 U/mL。重组菌株在含2%胆固醇的MRS培养基中培养48 h后,胆固醇降解率为94.43%。综上可知,本试验成功克隆了L.plantarum DPP8 BSH基因,并实现了其在L.casei CECT5276中的高效分泌表达。

本文引用格式

丁轲 , 余祖玲 , 王镨蒂 , 余祖华 , 李旺 , 李元晓 , 何万领 , 曹平华 , 张春杰 , 刘宁 . 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[J]. 动物营养学报, 2021 , 33(1) : 466 -473 . DOI: 10.3969/j.issn.1006-267x.2021.01.047

Abstract

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.

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