Construction of eukaryotic cell expression vectors pIRES2-EGFP-SP-B-C/T 1580 and evaluation of their expressions in 293T cells
10.3760/cma.j.issn.1008-1372.2012.10.013
- VernacularTitle:真核表达载体pIRES2-EGFP-SP-B-C/T 1580的构建及其在293T细胞中的表达
- Author:
Hongtao XU
;
Hui YANG
;
Liwen CHANG
;
Wenbin LI
;
Lingxia ZHAO
;
Wenhao YUAN
;
Wei LIU
;
Baohuan CAI
;
Xijuan WANG
- Publication Type:Journal Article
- Keywords:
Bronchopulmonary dysplasia/genetics;
Gene expression;
Genetic vectors;
Pulmonary surfactant-associated protein B/genetics
- From:
Journal of Chinese Physician
2012;(10):1338-1341
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct two kinds of eukaryotic ccll expression vcctors pIRES2-EGFP-SP-B-C/T 1580 and evaluate their expressions in 293T cells,for the further study of relationship between polymorphism of surfactant protein B (SP-B) gene and bronchopulmonary dysplasia (BPD).Methods The eukaryotic pIRES2-EGFP-SP-B-C/T 1580 expression vectors were constructed by gene recombination,and identified by gene sequencing.The recombinant expression vectors were transfected into 293T cells by lipofectamine2000.The expression of green fluorescence protein in 293T cells was observed by fluorescence microscopy.The mRNAs and proteins of SP-B-C/T 1580 were tested and identified by reverse transcriptionpolymerase chain reaction-restriction fragment length polymorphism(RT-PCR-RFLP) and western blot.Results Two recombinant plasmids contained the complete cDNA of SP-B with the same sequence as in gene bank.The base of SP-B 1580 gene of pIRES2-EGFP-SP-B-C 1580 was C,that of pIRES2-EGFP-SP-B-T 1580 was T.After being transfected into 293T cells,highly efficient expression of SP-B-C/T 1580 gene was detected at mRNA and protein levels.Conclusions The pIRES2-EGFP-SP-B-C/T 1580 eukaryotic cell expression vectors were successfully constructed.
