Construction and expression of bisbicistronic expression vector of novel endocrine and exocrine protein gene associated with breast cancer and IRES mediated gene EGFP.
- Author:
Huamei YAN
1
;
Yanping WANG
;
Yu WANG
;
Zhu WANG
;
Hong ZHENG
Author Information
1. Laboratory of Molecular Diagnosis of Cancer, State Key Laboratory of the Biotherapy and Cancer Center, West China Hospital , Sichuan University, Chengdu 610041, China.
- Publication Type:Journal Article
- MeSH:
Base Sequence;
Breast Neoplasms;
genetics;
pathology;
Female;
Genetic Vectors;
genetics;
Green Fluorescent Proteins;
biosynthesis;
genetics;
Humans;
MCF-7 Cells;
Molecular Sequence Data;
Proteins;
analysis;
genetics;
metabolism;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
Ribosomes;
chemistry;
metabolism
- From:
Journal of Biomedical Engineering
2012;29(4):737-744
- CountryChina
- Language:Chinese
-
Abstract:
This experimental study was aimed to construct the recombinant bisbicistronic eukaryotic expression vector containing endocrine and exocrine protein (EECP) gene associated with breast cancer and enhanced green fluorescent protein (EGFP) gene. And then we transfected it into breast cancer cells MCF-7 to detect the expression of EECP protein and study preliminary biological function of EECP gene. The EECP sequence was cloned to pBluescript II SK (+) plasmid. After restriction endonuclease reaction of pBluescript II SK(+) plasmid, the EECP fragment was cloned to pIRES2-EGFP vector forming a recombinant eukaryotic expression vector named pEECP-IRES2-EGFP. The potential vector was identified by restriction endonuclease digestion and sequencing. Correct plasmid was extracted and transfected into breast cancer cells MCF-7. The expression of EECP protein was detected by western blot analysis. Its biological function was studied by MTT and Flow-cytometry. It turns out that the recombinant eukaryotic expression vector containing EECP gene and EGFP gene was constructed successfully, and it could transfect MCF-7 cells efficiently. It can get higher expression of EECP protein and higher cell proliferation, thus providing an important and convenient tool for studying the function of EECP gene in vitro and in vivo.
