人特异性抗蛋白酶性保护因子Elafin的克隆及真核表达载体pEGFP-N1-elafin的构建及在肺上皮细胞中的表达

发布时间：2018-11-21 15:47

【摘要】：目的:克隆人特异性抗蛋白酶保护因子 Elafin 基因 cDNA,构建该基因的真核表达载体;观察其在肺上皮细胞中的表达情况,为研究Elafin 作为天然内生抗菌多肽的应用提供技术基础。方法:(1)抽提人肺总 RNA 进行逆转录反应,经 PCR 扩增目的片段,将其克隆于 pUCm-T 载体上,经酶切电泳、DNA 测序鉴定分析;定向亚克隆到绿色荧光蛋白表达载体 pEGFP-N1,并经酶切电泳、DNA 测序鉴定分析构建是否成功。(2)将 pEGFP-N1-elafin 转染到肺上皮细胞 A549 中,荧光显微镜观察其在细胞中表达及定位,并采用特异的cRNA 探针和抗 Elafin 单抗行 Northern 杂交和 ELISA 检测,分别从转录及蛋白质水平来分析其表达情况。结果:(1)经 RT-PCR 获得 377bp 的产物,经酶切、DNA 测序,确定所扩增片段为人 Elafin 基因 cDNA,进而成功筛选出表达载体pEGFP-N1-elafin。(2)转染后的肺上皮细胞中有 Elafin 基因表达,同时细胞培养上清液检测到 Elafin。结论:获得编码 Elafin 的基因片段,并成功构建了真核表达载体pEGFP-N1-elafin。在其转染的肺上皮细胞中有 Elafin 基因表达,且证实其主要是由培养细胞呈分泌性表达。Elafin 基因 cDNA 真核表达载体的成功构建以及主要呈分泌性表达的特点显示了极具意义的潜在应用价值。
[Abstract]:Aim: to construct the eukaryotic expression vector of human specific antiprotease protective factor (Elafin) gene cDNA, and observe its expression in pulmonary epithelial cells, and to provide a technical basis for the study of Elafin as a natural endophytic antibacterial peptide. Methods: (1) the total RNA of human lung was extracted for reverse transcription reaction. The target fragment was amplified by PCR and cloned into pUCm-T vector. It was identified by enzyme digestion electrophoresis and DNA sequencing. The expression vector of green fluorescent protein (pEGFP-N1,) was subcloned into GFP expression vector and analyzed by restriction endonuclease electrophoresis and DNA sequencing. (2) pEGFP-N1-elafin was transfected into lung epithelial cell line A549. Fluorescence microscopy was used to observe its expression and localization in cells. Northern hybridization and ELISA detection were performed with specific cRNA probe and anti Elafin monoclonal antibody to analyze its expression at the level of transcription and protein respectively. Results: (1) the product of 377bp was obtained by RT-PCR. The amplified fragment of human Elafin gene cDNA, was confirmed by restriction endonuclease digestion and DNA sequencing, and then the expression vector pEGFP-N1-elafin. (2) was successfully screened for Elafin gene expression in the transfected pulmonary epithelial cells. Elafin. was detected in the supernatant of cell culture at the same time. Conclusion: the gene fragment encoding Elafin was obtained and the eukaryotic expression vector pEGFP-N1-elafin. was successfully constructed. Elafin gene was expressed in the transfected lung epithelial cells. The successful construction of eukaryotic expression vector of Elafin gene cDNA and the characteristic of mainly secretory expression showed the potential value of application.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

【共引文献】

人特异性抗 蛋白酶性保护因子Elafin的克隆及真核表达载体pEGFP-N1-elafin的构建及在肺上皮细胞中的表达

人特异性抗蛋白酶性保护因子Elafin的克隆及真核表达载体pEGFP-N1-elafin的构建及在肺上皮细胞中的表达