[Construction of eukaryotic expression plasmid pEGFP-ATP1B1 and its effect on gastric adenocarcinoma cell SGC-7901].
OBJECTIVE
To establish the eukaryotic expression plasmid containing the code gene of Na+, K+-ATPase beta1-subunit (ATP1B1) and the basis of ATP1B1 applied to antitumor gene therapy.


METHODS
The ATP1B1 cDNA was amplified from leukocyte gene library and then cloned into the eukaryotic expression vector pEGFP-C3. The recombinant plasmid, named as pEGFP-ATP1B1, was determined with restriction enzyme and sequencing analyses. Next pEGFP-ATP1B1 was transferred into gastric adenocarcinoma SGC-7901 cells by lipofectamine, then ATP1B1 mRNA expression in transfected cells was detected by real-time PCR, and also ATPase was detected after cell transfection, as well as the proliferation of such cells was measured by MTT.


RESULTS
The analysis confirmed that the recombinant pEGFP-ATP1B1 contained the ATP1B1 cDNA. After cell transfection, the expression of ATP1B1 mRNA(129.2%) and the activity of ATPase [(2.95+/-0.210)%] were higher, and the growth of the SGC-7901 cells transfected with ATP1B1 was inhibited obviously when compared with the control group.


CONCLUSIONS
The recombinant pEGFP-ATP1B1 is constructed successfully, and this recombinant eukaryotic expression vector could be used in additional studies on the biological effect of ATP1B1 and its use in anti-tumor gene therapy.
Xiong
Zhu-Juan
ZJ
Division of Experimental Oncology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Lin
Ping
P
Zhang
Jie
J
Wang
Xiu-Jie
XJ
Wang
Qi
Q
Ren
Jing-Jing
JJ
Yang
Hong-Liang
HL
Wang
Jing
J
Wu
Ya-Ying
YY
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D001483:Base Sequence; D045744:Cell Line, Tumor; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014162:Transfection / Q000379:methods
C503551:ATP1B1 protein, human; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000254:Sodium-Potassium-Exchanging ATPase