Identification and verification of α-11 giardin-interacting protein
10.16250/j.32.1374.2022288
- VernacularTitle:α-11贾第素相互作用蛋白鉴定与验证
- Author:
Chenshuo ZHANG
1
;
Lei HUANG
2
;
Yu TANG
1
;
Peng WANG
1
;
Yalan CHEN
1
;
Liu ZHANG
1
;
Hai’e SHEN
1
;
Yuan YU
1
;
Xifeng TIAN
1
;
Yang WANG
1
Author Information
1. College of Life Sciences, North China University of Technology, Tangshan, Hebei 063000, China
2. Hongci Hospital of Tangshan City, Hebei Province, China
- Publication Type:Journal Article
- Keywords:
Giardia lambia;
α-11 giardin;
Protein-protein interaction;
Yeast two-hybridization;
Bimolecular fluorescence complementation
- From:
Chinese Journal of Schistosomiasis Control
2023;35(2):155-162
- CountryChina
- Language:Chinese
-
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.
