hGPR177/PCDNA3.1(+)重组质粒的构建及其在A549肺癌细胞中的表达

Reconstruction of hGPR177/PCDNA3.1(+) plasmid and expression of hGPR177 protein in A549 lung cancer cells

目的构建hGPR177/PCDNA3.1(+)重组质粒,并在A549肺癌细胞中表达hGPR177蛋白。方法利用生物分析软件DNAMAN6.0选取合适的穿梭载体PCDNA3.1(+)和酶切位点。用质粒提取试剂盒对穿梭载体PCDNA3.1(+)及含有hGPR177基因pReceive-B04质粒进行抽提,并对其进行双酶切。使用DNA连接酶把hGPR177基因与PCDNA3.1(+)酶切片段连接,重新构建hGPR177/PCDNA3.1(+)重组质粒。将重组质粒通过电转的方式转导进A549肺癌细胞。运用细胞培养技术,培养已电转hGPR177/PCDNA3.1(+)重组质粒的A549肺癌细胞。结果利用0.8%琼脂糖凝胶电泳检测hGPR177/PCDNA3.1(+)重组质粒的质量为6999bp。经过机械破碎后获取细胞提取物,电泳鉴定出现GPR177蛋白带。结论成功构建了hGPR177/PCDNA3.1(+)重组质粒,并在A549肺癌细胞表达系统中表达出人GPR177蛋白。

Objective To construct the hGPR177/PCDNA3.1（＋） recombinant plasmid and to express the hGPR177 protein in A549 lung cancer cells. Methods A suitable shuttle vector PCDNA3.1 （＋） and restriction sites were selected by bio-analysis software DNAMAN6.0. A shuttle vector PCDNA3.1 （＋） and pReceive-B04 plasmid with hGPR177 gene were extracted by the plasmid extraction kit and then was treated by end nucleases digesting, hGPR177 gene and PCDNA3.1 （＋） fragments were recombined by using DNA ligase, and hGPR177/PCDNA3.1 （＋） recombinant plasmid was reconstructed. The recombinant plasmids were transferred into A549 lung cancer cells electrical transduction. The A549 lung cancer cells with electrotransfection of GPR177/PCDNA3.1 （＋） recombinant plasmid was trained by using cell culture technology. Results The quality of the hGPR177/PCDNA3.1（＋） recombinant plasmid was 6999 bp. Electrophoresis revealed hGPR177 protein in the broken cell extracts. Conclusion A549 lung cancer cells containing hGPR177/PCDNA3.1（＋） recombinant plasmid can express hGPR177 protein.