Construction of LAT1 eukaryotic expression vector of C57 mouse and its effect on the Neuro-2a cell
10.3969.j.issn.1671.7856.2014.005.006
- VernacularTitle:C57小鼠 LATl 真核表达载体的构建及其对Neuro-2a 细胞的影响
- Author:
Bingyan WEI
;
Tianfu LIU
;
Linhua FAN
;
Maolin LIU
- Publication Type:Journal Article
- Keywords:
Lamino acid transporter 1;
Eukaryotic expression vector;
Neuro-2a cell;
Proliferation;
Apoptosis
- From:
Chinese Journal of Comparative Medicine
2014;(5):25-30,9
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the lamino acid transporter 1 eukaryotic expression vector of C 57 mouse and to express the gene inNeuro-2atumor cells,and explore the effect of LAT1on proliferation and apoptosis of Neuro-2a cell. Methods The full-length LAT1 cDNA was synthesized by RT-PCR and cloned into pcDNA3.1vector to construct recombinant plasmid.The constructed pcDNA3.1-LAT1vector was verified by Enzyme digestion and sequencing and then transfected intomurine Neuro-2acellsby liposome.The transfected cells were selected with G418 and stably expressed strain was constructed .The expression of LAT1 was detected by RT-PCR and western blot .Proliferation was analyzed by MTT , cell cycle and apoptosis were detected by flow cytometric analysis .Results The full-length LAT1 cDNA was amplified successfully and pcDNA3.1-LAT1eukaryoticvector was constructed successfully .Enzyme digestion and sequencing confirmed the sequence was correct .Neuro-2acells were transfected and Stably expressed strain was constructed successfully.MTT showed that the group of transfected restructuring plasmid could significantly affect Neuro -2a cell proliferation more than the control groups ( P <0.05 ) .From the flowcytometric analysis , LAT1 could promote cell proliferation and inhibit Neuro-2a cell apoptosis.Conclusion LAT1 can express successfully inNeuro-2acells which were transfected with recombinantpcDNA3.1-LAT1plasmid.LAT1 in Neuro-2a cells can promote cell proliferation and inhibit the cell apoptosis which provides a basis for the study of LAT 1.That lays the foundation for studying biological effects of LAT 1.
