Construction and expression of the eukaryotic expression vector of wild-type p27 and lacking the nuclear localization signal p27

Xinyan Jiao; Ying Zhang; Qiu Sheng; Miao Zhang; Zejian Yang; Xiaoqian Gao; Qian Zhao; Bo Wang; Peijun Liu

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Construction and expression of the eukaryotic expression vector of wild-type p27 and lacking the nuclear localization signal p27

VernacularTitle:野生型p27和核定位信号缺失型p27真核表达载体的构建及表达
Author: Xinyan JIAO ^{1
,

2} ; Ying ZHANG ³ ; Qiu SHENG ⁴ ; Miao ZHANG ^{1
,

2} ; Zejian YANG ^{1
,

2} ; Xiaoqian GAO ^{1
,

2} ; Qian ZHAO ⁵ ; Bo WANG ^{1
,

2} ; 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. Department of Gastroenterology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061
4. Hospital of Northwestern Polytechnical University, Xi’an 710072
5. Department of Otolaryngology‒Head and Neck Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China
Publication Type:Journal Article
Keywords: p27; CDKN1B; nuclear localization signal; eukaryotic expression
From: Journal of Xi'an Jiaotong University(Medical Sciences) 2022;43(4):516-521
CountryChina
Language:Chinese
Abstract: 【Objective】 To construct the eukaryotic expression vector carrying the human wild-type p27 and lacking nuclear localization signal p27△NLS coding sequences， and the express them in HEK293T cells， which may contribute to investigating the different locations and roles of p27 in the cytoplasm and nucleus. 【Methods】 Total RNA was prepared from human breast cancer MCF7 cells， and cDNA was obtained by reverse transcription-polymerase chain reaction （RT-PCR）. After amplification of the p27 CDs and non-NLS fragments by PCR， full length p27WT （CDKN1B， NM_004064.5） and p27△NLS coding regions were obtained. PCR products were then subcloned into the eukaryotic expression vector pCMV-Blank. After identification with bacterial PCR， double restriction enzyme digestion and sequencing， they were defined officially as pCMV-p27WT and pCMV-p27△NLS， respectively. The recombinant plasmids were transfected into HEK293T cells by electroporation. After 48 h， the levels of p27 protein in the cytoplasm and nucleus were detected by Western blotting. 【Results】 The sequencing results showed that the sequences of p27WT and p27△NLS inserted into the plasmids were both correctly consistent with that of NM_004064.5. After transfection with pCMV-p27WT， total p27 protein expression was increased and distributed in both the cytoplasm and nucleus of HEK293T cells. After transfection with pCMV-p27△NLS， p27 protein was significantly increased and almost entirely localized in the cytoplasm of HEK293T cells. 【Conclusion】 The eukaryotic expression plasmids of human p27WT and p27△NLS coding sequences were successfully constructed and overexpressed in HEK293T cells. This research may lay a foundation for investigating the biological function of p27 in the cell cycle progression of tumor cells.