摘要
目的构建含人SLC2A9基因的过表达慢病毒载体,探讨其对人肾脏近曲小管上皮细胞株HK-2尿酸转运功能的影响。方法 PCR反应扩增目的基因后连入慢病毒表达载体中构建重组载体pLEX-SLC2A9,使用PCR及测序的方法对其进行鉴定,并与辅助包装质粒共感染293T细胞。慢病毒颗粒感染HK-2细胞后,用PCR和Western blot检测SLC2A9基因的过表达效率,并检测SLC2A9过表达对其尿酸转运功能的影响。结果 PCR及测序结果表明重组慢病毒载体pLEX-SLC2A9的插入序列完全正确,重组慢病毒载体感染HK-2细胞后,细胞内的mRNA及蛋白水平增高,并且可以增强HK-2细胞对尿酸的转运。结论过表达SLC2A9基因后能显著增强HK-2细胞对尿酸的转运。
Objective To construct lentiviral vector over expressing human SLC2A9 gene, and to investigate its effect on uric acid transport function of HK-2 cells. Methods Human SLC2A9 gene was amplified by polymerase chain reaction. Gene amplification products were inserted into the lentiviral vector pLEX, and lentiviral vector pLEX-SLC2A9 was constructed. The constructed vector was verified by PCR analysis and DNA sequencing. Lentiviral vector pLEX-SLC2A9 and virus packaging plasmids were cotransfected into 293T cells, and then transfected into human kidney HK-2 cells. PCR and Western blotting were used to determine the transfecting efficiency of SLC2A9 gene, and the effect of SLC2A9 gene over expression on uric acid transport in HK-2 cells was observed. Results PCR analysis and DNA sequencing showed that recombinant lentivirus plas- mids pLEX-SLC2A9 were constructed successfully. In transfected HK-2 cells, SLC2A9 gene mRNA and protein were over-ex- pressed. Compare to normal HK-2 cells, transfected cells had stronger uric acid transport activity. Conclusion Overexpres- sion of SLC2A9 gene can significantly increase the uric acid transport function of HK-2 cells.
出处
《现代泌尿外科杂志》
CAS
2014年第8期531-535,共5页
Journal of Modern Urology
基金
国家自然科学基金面上项目(No.81370804)
广州市属高校羊城学者科研项目(No.12A017S)
