中国血吸虫病防治杂志 ›› 2023, Vol. 35 ›› Issue (2): 155-.

• 论著 • 上一篇    下一篇

α⁃11贾第素相互作用蛋白鉴定与验证

张晨烁1,黄磊2,唐瑜1,王朋1,陈亚澜1,张柳1,沈海娥1,余源1,田喜凤1,王洋1*   

  1. 1 华北理工大学生命科学学院(河北 唐山 063000); 2 河北省唐山市弘慈医院
  • 出版日期:2023-04-15 发布日期:2023-05-19
  • 作者简介:张晨烁,男,硕士研究生。研究方向:寄生虫学、植物与合成生物学
  • 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(X2021079)

Identification and verification of α⁃11 giardin⁃interacting protein

ZHANG Chenshuo1, HUANG Lei2, TANG Yu1, WANG Peng1, CHEN Yalan1, ZHANG Liu1, SHEN Hai’e1, YU Yuan1, TIAN Xifeng1, WANG Yang1*   

  1. 1 College of Life Sciences, North China University of Technology, Tangshan, Hebei 063000, China; 2 Hongci Hospital of Tangshan City, Hebei Province, China
  • Online:2023-04-15 Published:2023-05-19

摘要: 目的 鉴定并验证α⁃11贾第素的相互作用蛋白,为其功能研究提供实验依据。方法 构建C2株蓝氏贾第鞭毛虫酵母双杂交cDNA文库和α⁃11贾第素诱饵质粒,通过酵母双杂交筛选出可能与α⁃11贾第素相互作用的蛋白。将α⁃11贾第素和筛选出的可能互作蛋白基因分别构建入pBiFc⁃Vc⁃155、pBiFc⁃Vn⁃173质粒,共转染乳腺癌细胞系MDA⁃MB⁃231,通过双分子荧光互补(bimolecular fluorescent complementation, BiFC)实验验证两种蛋白的相互作用。结果 构建了库容为2.715 × 107 细菌菌落总数(CFU)的蓝氏贾第鞭毛虫酵母双杂交cDNA文库和含有α⁃11贾第素基因的无自激活活性的诱饵质粒。通过酵母双杂交两轮阳性筛选,共筛选出6个可能与α⁃11贾第素相互作用的蛋白,分别为真核翻译起始因子5A(eukaryotic translation initiation factor 5A,EIF5A)、钙调蛋白激酶(calmodulin⁃dependent protein kinase,CAMKL)、烟酰胺腺嘌呤二核苷酸磷酸特异性谷氨酸脱氢酶(NADP⁃specific glutamate dehydrogenase, NADP⁃GDH)、假设蛋白1(GL50803_95880)、假设蛋白2 (GL50803_87261)和一段来自犬贾第虫病毒的蛋白。成功将α⁃11贾第素基因与EIF5A基因分别转入BiFc系统质粒pBiFc⁃Vc⁃155与pBiFc⁃Vn⁃173,将矫正突变的pBiFc⁃Vc⁃155⁃α⁃11与pBiFc⁃Vn⁃173⁃EIF5A重组质粒共转染MDA⁃MB⁃231细胞后,镜下观察可见绿色荧光,证实α⁃11贾第素与EIF5A蛋白在细胞内存在相互作用。结论 成功构建了C2株蓝氏贾第鞭毛虫酵母双杂交文库,并鉴定出6个α⁃11贾第素可能的相互作用蛋白,其中EIF5A与α⁃11贾第素在细胞内存在相互作用。  

关键词: 蓝氏贾第鞭毛虫, α?11贾第素, 蛋白相互作用, 酵母双杂交, 双分子荧光互补

Abstract: Objective To identify and verify the interacting protein of α⁃11 giardin, so as provide the experimental evidence for studies on the α⁃11 giardin function. Methods The yeast two⁃hybrid cDNA library of the Giardia lambia C2 strain and the bait plasmid of α⁃11 giardin were constructed. All proteins interacting with α⁃11 giardin were screened using the yeast two⁃hybrid system. α⁃11 giardin and all screened potential interacting protein genes were constructed into pBiFc⁃Vc⁃155 and pBiFc⁃Vn⁃173 plasmids, and co⁃transfected into the breast cancer cell line MDA⁃MB⁃231. The interactions between α⁃11 giardin and interacting proteins were verified using bimolecular fluorescence complementation (BiFC). Results The yeast two⁃hybrid G. lambia cDNA library which was quantified at 2.715 × 107 colony⁃forming units (CFU) and the bait plasmid containing α⁃11 giardin gene without an autoactivation activity were constructed. Following two⁃round positive screening with the yeast two⁃hybrid system, two potential proteins interacting with α⁃11 giardin were screened, including eukaryotic translation initiation factor 5A (EIF5A), calmodulin⁃dependent protein kinase (CAMKL) and nicotinamide adenine dinucleotide phosphate⁃specific glutamate dehydrogenase (NADP⁃GDH), hypothetical protein 1 (GL50803_95880), hypothetical protein 2 (GL50803_87261) and a protein from Giardia canis virus. The α⁃11 giardin and EIF5A genes were transfected into the pBiFc⁃Vc⁃155 and pBiFc⁃Vn⁃173 plasmids using BiFC, and the recombinant plasmids pBiFc⁃Vc⁃155⁃α⁃11 and pBiFc⁃Vn⁃173⁃EIF5A were co⁃tranfected into MDA⁃MB⁃231 cells, which displayed green fluorescence under a microscope, indicating the interaction between α⁃11 giardin and EIF5A protein in cells. Conclusion The yeast two⁃hybrid cDNA library of the G. lambia C2 strain has been successfully constructed, and six potential protein interacting with α⁃11 giardin have been identified, including EIF5A that interacts with α⁃11 giardin in cells.

Key words: Giardia lambia, α?11 giardin, Protein?protein interaction, Yeast two?hybridization, Bimolecular fluorescence complementation

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