[Construction of expression vector of hTERT/hIL-18 fusion gene in eukaryotic cells and its function].
To construct expression vector of hTERT-hIL-18 fusion gene in eukaryotic cells and to study its biological function.



hIL-18 gene was amplified by RT-PCR, then T-A cloned and inserted into PCDNA3.1(+)/hTERT vector. The sequence of fusion gene was examined by enzyme incision and DNA sequencing. The vector with fusion gene was transformed into 3T3 cells by the method of lipofecting, and proved by Western blot. The secretion gamma-interferon was measured with ELISA and cell apoptosis was detected with flow cytometry.



Expression vector PCDNA3.1(+) of hTERT/hIL-18 fusion gene was constructed successfully. The correct sequence was proved by enzyme incision and sequencing and there was a correct open reading frame. Fusion protein of hTERT/hIL-18 was effectively expressed in eukaryotic cells and was proved by Western blot and immunofluorescence stain. The fusion protein stimulated KG-1 cells to secrete gamma-interferon and had anti-apoptosis effect.



Fusion protein hTERT-hIL-18 is highly effectively expressed in eukaryotic cells and is biologically active.
Tong
Xiang-min
XM
Department of Hematology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Jin
Jie
J
Yao
Hang-ping
HP
Qian
Wen-bin
WB
Qian
Hai-tao
HT
D001483:Base Sequence; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D020382:Interleukin-18 / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D019098:Telomerase / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
D020382:Interleukin-18; D011993:Recombinant Fusion Proteins; C509186:TERT protein, human; D019098:Telomerase