人端粒酶逆转录酶和人白细胞介素18融合基因真核表达载体的构建和功能研究

Construction of expression vector of hTERT/hIL-18 fusion gene in eukaryotic cells and its function

目的:以基因重组技术在真核细胞中表达人白细胞介素18(hIL-18)和人端粒酶逆转录酶(hTERT)融合基因,并观察其生物学功能。方法:健康人单核细胞经RT-PCR扩增hIL-18后、T-A克隆。亚克隆至经N heⅠ、H indⅢ酶切的PCDNA 3.1(+)/hTERT中,限制性内切酶验证后,挑选出含有目标基因的正确克隆。经限制性内切酶和DNA测序分析两个基因是否已经正确连接到真核表达载体中。将hTERT/hIL-18融合基因质粒脂质体介导转染3T 3细胞中表达,经W estern-b lot验证目的基因表达产物,同时对转染细胞作免疫荧光染色。以EL ISA检测转染细胞刺激KG-1细胞分泌γ干扰素的能力,以流式细胞仪检测转染细胞抗MTX诱导凋亡的能力。结果:hTERT/hIL-18融合基因的真核表达载体PCDNA 3.1(+)构建成功,经限制性内切酶验证和DNA测序及同源性对比分析,该基因片段与NCB I基因库hIL-18和hTERT基因CDS序列同源性均达到99.9%;而细胞转染后经W estern-b lot验证,hTERT/hIL-18融合蛋白的分子量约127 kM r,与预期一致,荧光显微镜观察显示融合蛋白在细胞中弥散表达。此融合蛋白能刺激KG-1细胞分泌γ-干扰素,并具有抗MTX诱导凋亡的生物学功能。结论:本实验构建的PCDNA 3.1(+)/hTERT-hIL-18质粒能够在真核细胞中表达,并具有生物学功能,为进一步转染树突状细胞,研制肿瘤的治疗性疫苗提供基础。

Objective： To construct expression vector of hTERT-hIL-18 fusion gene in eukaryotic cells and to study its biological function. Methods： 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 γ-interferon was measured with ELISA and cell apoptosis was detected with flow cytometry. Results： 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 γ-interferon and had anti-apoptosis effect. Conclusion： Fusion protein hTERT-hIL-18 is highly effectively expressed in eukaryotic cells and is biologically active.