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目的构建Endocan真核表达载体pcDNA3.1-En-docan,转染犬肾小管上皮细胞MDCK,观察其在MDCK中的表达并初步分析其对骨桥蛋白(OPN)表达的影响。方法应用PCR从pET28-Endocan中扩增出Endocan cDNA全长序列,酶切后导入pcDNA 3.1/myc-his A多克隆位点,构建真核表达载体pcDNA3.1-Endocan;瞬时转染MDCK细胞,Western blot检测Endocan和OPN表达。结果经双酶切鉴定及测序分析表明Endocan已经克隆到pcDNA 3.1/myc-his A中。Western blot结果表明转染成功,Endocan表达量增加,且OPN随其而增加。结论Endocan可促进MDCK表达OPN。
Objective To construct Endocan eukaryotic expression vector pcDNA3.1-En-docan and transfect MDCK in renal tubular epithelial cells to observe its expression in MDCK and to analyze its effect on osteopontin (OPN) expression. Methods Endocan cDNA was amplified from pET28-Endocan by PCR and inserted into pcDNA3.1 / myc-his A multi-cloning site to construct eukaryotic expression vector pcDNA3.1-Endocan. Transient transfection of MDCK cells, Western blot detection of Endocan and OPN expression. Results Double enzyme digestion and sequencing analysis showed that Endocan had been cloned into pcDNA 3.1 / myc-his A. Western blot results showed that transfection was successful, Endocan expression increased, and OPN increased with it. Conclusion Endocan can promote MDCK expression of OPN.