Construction of expression vector of hTERT/hIL-18 fusion gene in eukaryotic cells and its function / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To construct expression vector of hTERT-hIL-18 fusion gene in eukaryotic cells and to study its biological function.</p><p><b>METHODS</b>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.</p><p><b>RESULT</b>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.</p><p><b>CONCLUSION</b>Fusion protein hTERT-hIL-18 is highly effectively expressed in eukaryotic cells and is biologically active.</p>