Construction of eukaryotic expression vector and bioinformatics analysis of human kidney and brain protein (KIBRA)

Bo Wang; Shaoran Song; Bixia Tian; Zejian Yang; Miao Zhang; Xiaoqian Gao; Wei Sun; Yina Jiang; Peijun Liu

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Construction of eukaryotic expression vector and bioinformatics analysis of human kidney and brain protein (KIBRA)

VernacularTitle:人KIBRA基因真核表达载体的构建及生物信息学分析
Author: Bo WANG ^{1
,

2} ; Shaoran SONG ^{1
,

2} ; Bixia TIAN ^{1
,

2} ; Zejian YANG ^{1
,

3} ; Miao ZHANG ^{1
,

2} ; Xiaoqian GAO ^{1
,

2} ; Wei SUN ^{1
,

2} ; Yina JIANG ⁴ ; Peijun LIU ^{1
,

2}
Author Information

1. Center for Translational Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061
2. Key Laboratory for Tumor Precision Medicine of Shaanxi Province, Xi’an 710061
3. Xi’an Jiaotong University Health Science Center, Xi’an 710061
4. Department of Pathology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China
Publication Type:Journal Article
Keywords: KIBRA; eukaryotic expression; gene clone
From: Journal of Xi'an Jiaotong University(Medical Sciences) 2021;42(2):323-327,332
CountryChina
Language:Chinese
Abstract: 【Objective】 To clone the full-length of human kidney and brain protein (KIBRA) coding sequence in eukaryotic expression vector and provide a model for studying the biological function of KIBRA in breast cancer cells. 【Methods】 Total RNA of human breast cancer cell line MCF7 was extracted. After reverse transcription, the full length of KIBRA (NM_001161661.2) coding region was amplified by PCR, and cloned into eukaryotic expression vector pCMV-Blank. After identification, it was defined officially as pCMV-KIBRA. Then it was transfected into MCF7 cells, and the expression of KIBRA was detected by real-time PCR and Western blotting after 48 hours. The primary, secondary and tertiary structures and post-transcriptional modification sites of KIBRA were analyzed with bioinformatics software. 【Results】 Bacterial PCR, double enzyme digestion and DNA sequencing results showed that the correct sequence of KIBRA was inserted into the vector pCMV-KIBRA. The mRNA and protein expressions of KIBRA were significantly increased in MCF7 cells transfected with pCMV-KIBRA. Bioinformatics analysis showed that KIBRA was composed of 1119 amino acids. There were 52 phosphorylation sites, 1 acetylation site and 5 ubiquitination sites, and the protein structure was mainly α-helix and random coil. 【Conclusion】 The eukaryotic expression vector of full-length of human KIBRA coding sequence was successfully constructed and overexpressed in breast cancer cell line MCF7, which can lay a foundation for studying the biological function of KIBRA in breast cancer.