热带病与寄生虫学

热带病与寄生虫学 ›› 2020, Vol. 18 ›› Issue (1): 21-24.

• 论著 • 上一篇    下一篇

蓝氏贾第鞭毛虫末端结合蛋白 1 基因的克隆与原核表达

李汶霖1 , 王鸿斌2 , 李淑凝1 , 王洋2∗ , 李志敏3 , 沈海娥2 , 刘红宁2 , 李幽美2 , 赵旭安1   

  1. 1. 华北理工大学临床医学院,唐山 063000; 2. 华北理工大学生命科学学院; 3. 华北理工大学护理与康复学院
  • 收稿日期:2019-12-20 出版日期:2020-03-10 发布日期:2020-03-10
  • 通信作者: ∗通讯作者,王洋,E-mail:konig718@163.com
  • 作者简介:李汶霖,男,本科,研究方向:临床医学。 E-mail:1044735985@qq.com
  • 基金资助:
    河北省自然科学基金 (H2017209143);华北理工大学大学生创新创业项目(X2019181)

Cloning and prokaryotic expression of Giardia lamblia eb1 gene

LI Wen-lin1 ,WANG Hong-bin2 ,LI Shu-ning1 , WANG Yang2∗ , LI Zhi-min3 , SHEN Hai-e2 , LIU Hong-ning2 , LI You- mei2 , ZHAO Xu-an1   

  1. 1. College of Clinical Medicine, North China University of Science and Technology, Tangshan 063210, China; 2. College of Life Sci- ences,North China University of Science and Technology, 3. College of Nursing and Rehabilitation, North China University of Science and Technology,
  • Received:2019-12-20 Online:2020-03-10 Published:2020-03-10
  • Contact: ∗ Corresponding author,E-mail:konig718@163.com

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摘要： 目的 克隆并原核表达 C2 株蓝氏贾第鞭毛虫(Giardia lambia,简称贾第虫)末端结合蛋白 1(End-bind- ing protein 1,geb1)基因,获得重组 gEB1 蛋白。 方法 由于 geb1 基因无内含子,我们以 C2 株贾第虫基因组为模板,以国际标准株WB 株geb1 基因序列为参考序列,设计引物克隆 geb1 基因,经 NcoⅠ和 XhoⅠ双酶切与原核表达载体 pET-28α(+)连接,转化感受态 E. coli TOP10,经筛选和测序验证后导入大肠杆菌 E. coli Rosetta(DE3),异丙基-β-D -硫代半乳糖(IPTG)诱导 gEB1 蛋白表达,产物经 SDS-PAGE 和 Western blot 进行检测和验证。 结果 成功构建了表 达 C2 株贾第虫 geb1 基因的原核表达载体 pET-28α(+)- gEB1,转化入大肠杆菌 E. coli Rosetta(DE3),重组菌株经 0. 1 mmol / L IPTG ,30℃低温诱导 5 h, SDS-PAGE 和 Western blot 显示,在相对分子量约 29 KDa 的位置出现目的蛋 白条带,与理论值一致。 结论 用大肠杆菌成功表达了 gEB1 蛋白,为 gEB1 蛋白的功能研究和抗体制备提供了材料。

关键词: 蓝氏贾第鞭毛虫, 末端结合蛋白 1, 原核表达

Abstract: Objective To clone and express the C2 strain of Giardia lambia End-binding protein 1(gEB1) in E. coli. Methods Recombinant gEB1 protein was obtained, on which basis the primer was designed to clone gEB1 gene using C2 strain of Giardia lamblia as template and sequence reference to gEB1 gene by international standard WB strain The PCR product was cloned into prokaryotic expression vector pE-28α(+) with restriction enzymes Nco I and Xho I. The recombinant vector pET-28a(+)-gEB1 was transformed into E. coli host strain Rosetta(DE3), then the fusion protein was expressed by IPTG induction at 30℃ for 5 h. All products were tested and confirmed by SDS-PAGE and Western blot. Results The prokaryotic expression vector was successfully constructed, and the EB1 was highly expressed in E. coli Rosetta (DE3). SDS-PAGE and Western blot showed that the expressed product was about 29 kDa that was consistent with the defined value. Conclusion gEB1 gene has been successfully cloned and expressed in E. coli Rosetta(DE3), the recombinant protein can be used for further functional study and antibody preparation.

Key words: Giardia lambia, End-binding protein 1, Prokaryotic expression

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