上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2018, Vol. 38 ›› Issue (11): 1306-.doi: 10.3969/j.issn.1674-8115.2018.11.006

• 论著·基础研究 • 上一篇    下一篇

大肠埃希菌CLM37菌株Lpp基因敲除及胞外表达N-糖基化重组蛋白研究

阮瑶，王力凡，郭龙华，付鑫，丁宁 ，胡学军   

  1. 大连大学医学研究中心，大连 116622
  • 出版日期:2018-11-28 发布日期:2018-12-15
  • 通讯作者: 丁宁，电子信箱：dingning0606@foxmail.com。胡学军，电子信箱：xuejun.hu3@foxmail.com。为共同通信作者。
  • 作者简介:阮瑶（1990—），女，硕士生；电子信箱： ruanyao17@foxmail.com。
  • 基金资助:
    国家自然科学基金（31370937）

Knocking out Lpp gene of E.coli CLM37 strain and extracellular production of N-glycosylated recombinant proteins

RUAN Yao, WANG Li-fan, GUO Long-hua, FU Xin, DING Ning, HU Xue-jun   

  1. Medical Research Centre, Dalian University, Dalian 116622, China
  • Online:2018-11-28 Published:2018-12-15
  • Supported by:
    National Natural Science Foundation of China, 31370937)。

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摘要： 目的 ·构建外膜脂蛋白 Lpp（Brauns lipoprotein）基因缺失的大肠埃希菌菌株，在该菌株中进行大肠埃希菌胞外生产 N-糖基化重组蛋白研究。方法 ·利用 Red同源重组系统，敲除大肠埃希菌 CLM37基因组上的外膜脂蛋白 Lpp基因；通过绘制生长曲线，研究 Lpp基因缺失后对大肠埃希菌生长状态的影响。将受体蛋白表达载体 pIG6-rFn3-Gly和空肠弯曲杆菌来源的 N-糖基化基因簇载体 pACYCpgl共转化大肠埃希菌 CLM37 Δ Lpp，研究大肠埃希菌胞外生产 N-糖基化重组蛋白情况。结果 ·获得了 Lpp基因缺失的大肠埃希菌菌株 CLM37 Δ Lpp，并在该菌株中成功实现了胞外生产 N-糖基化重组蛋白 rFn3-Gly；相比于菌株 CLM37，大肠埃希菌 CLM37 Δ Lpp胞外生产重组蛋白 rFn3-Gly的总量约提升了 4倍，糖基化效率约提高了 6倍。结论 ·成功构建大肠埃希菌菌株 CLM37 Δ Lpp并实现了胞外生产 N-糖基化重组蛋白 rFn3-Gly，提高了糖蛋白产量及糖基化效率。

关键词: Lpp基因, 大肠埃希菌 CLM37, N-糖基化蛋白

Abstract:

Objective · To construct the E.coli CLM37 strain with Lpp gene deletion and to study the production of N-glycosylated recombinant proteins in this E.coli strain. Methods · Firstly, Red homologous recombination system was used to knock out the Lpp gene the genome of E.coli CLM37. And then, the growth curve was detected to study the effects of deleted Lpp gene on the growth states of E.coli strain. Finally, the vector pIG6-rFn3Gly which expresses receptor protein and the vector pACYCpgl, which carries N-glycosylation gene cluster that derives Campylobacter jejuni, were co-transformed into E.coli CLM37ΔLpp to investigate the extracellular production of N-glycosylated recombinant proteins. Results · The E. coli CLM37ΔLpp with Lpp gene deletion was obtained, and the extracellular production of N-glycosylated rFn3-Gly was successfully achieved in this strain. Compared with E. coli CLM37, the total amount of rFn3-Gly produced via extracellular production of E. coli CLM37ΔLpp increased about 4 times, and the glycosylation efficiency increased about 6 times. Conclusion · N-glycosylated rFn3-Gly was successfully produced via extracellular production in E. coli CLM37ΔLpp, and the production of interest glycoprotein and the glycosylation efficiency were improved.

Key words: Lpp gene, E.coli CLM37, N-glycoprotein

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