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野生型犬尿氨酸酶表达载体的构建及酶活性检测

Construction of wild kynureninase expression vector and determination of kynureninase activity
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摘要 目的构建野生型犬尿氨酸酶(KYNU)的真核表达载体,并分析其在HEK293细胞中的表达及其酶活性。方法抽提人肝脏细胞的总RNA,通过反转录聚合酶链反应(RT-PCR)法获得KYNU基因全长cDNA,将野生型KYNU基因克隆到pcDNA载体质粒中,获得野生型pcDNA-KYNU重组表达质粒,经酶切鉴定和测序验证后转染HEK293细胞,用蛋白质印迹法技术检测野生型KYNU在细胞中的表达,用高效液相色谱法(HPLC)检测HEK293细胞表达的野生型KYNU酶蛋白的活性。结果通过测序及酶切鉴定野生型pcDNAKYNU重组表达质粒扩增后的PCR产物电泳结果显示构建成功,蛋白质印迹法结果显示转染有重组野生型cDNA-KYNU质粒的HEK293细胞能表达KYNU蛋白,HPLC结果显示野生型KYNU酶存在活性。结论成功构建野生型pcDNA-KYNU的真核表达载体,野生型重组质粒在HEK293细胞中能够表达KYNU并具有一定的酶活性。 Objective To construct the eukaryotic expression vector of wild kynureninase (KYNU), and to de-termine its expression and enzymatic activity in HEK293 cells. Methods The total RNA of human hepatic cells was extracted. The full-length cDNA of KYNU gene was obtained by RT-PCR to obtain the wild-type pcDNA-KYNU re-combinant expression plasmid. After measuring by restriction enzyme digestion and sequencing, the wild-type KYNU recombinant expression plasmid was transfected into HEK293 cells. The expression of wild-type KYNU was deter-mined by Western blot. The activity of wild-type KYNU enzyme protein expressed in HEK293 cells was determined by high performance liquid chromatography (HPLC). Results The restriction enzyme digestion and sequencing veri-fied that the PCR products of electrophoresis were successfully constructed after amplification of the wild-type pcD-NA-KYNU recombinant expression plasmid. Western blot showed that the HEK293 cells transfected with recombinant wild-type cDNA-KYNU plasmid could express KYNU protein. HPLC showed that the wild-type KYNU enzyme was active. Conclusion The wild-type pcDNA-KYNU eukaryotic expression vector was constructed successfully. The wild-type recombinant plasmid could express KYNU in HEK293 cells, which had a certain enzyme activity.
出处 《中国药物与临床》 CAS 2017年第4期465-467,I0001,共4页 Chinese Remedies & Clinics
基金 国家自然科学基金(30270545) 浙江省教育厅科研项目(Y201017610) 浙江省中医药普通项目(2009CB048)
关键词 犬尿氨酸酶 野生型 高血压 高效液相色谱法 Kynureninase Wild-type Hypertension High performance liquid chromatography
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