高血压大鼠SLC7A8基因腺病毒载体的构建及鉴定

摘要

目的 构建携带SLC7A8基因的重组腺病毒载体,为进一步研究SLC7A8基因的功能作准备.方法 含SLC7A8目的 基因编码序列(coding domain sequence,CDS)的互补DNA(complementary DNA,cDNA)的pGEM-T Easy载体测序分析正确后,再将其连接入重组腺病毒载体.以双酶切和测序鉴定正确后,将pAdxsi-GFP-SLC7A8重组腺病毒载体转染大鼠肾小管上皮细胞(rat renal tubular epithelial cells-52e,NRK-52E),流式细胞仪测定转染效率,反转录聚合酶链反应法检测转染后SLC7A8 mRNA的表达.结果 pAdxsi- GFP-SLC7A8重组腺病毒载体构建成功,流式细胞仪测定重组腺病毒载体转染NRK-52E效率为94.1%,且证实转染后肾小管上皮细胞的SLC7A8 mRNA表达.结论 本研究成功构建pAdxsi-GFP-SLC7A8重组腺病毒载体,并成功转染NRK-52E细胞,这为我们进一步探索SLC7A8基因的生物学功能奠定了基础.%Objective To construct the recombinant adenoviral vector with rat SLC7 A8 gene for its function study in spontaneously hypertensive rat. Methods The pGEM - T Easy vector which contained SLC7A8 complementary DNA( cDNA ) was confirmed by sequence analysis. Then the pGEM T Easy vector which contained SLC7A8 cDNA was cloned into the recombinant adenoviral vector and the recombinant adenoviral vector was confirmed by double enzyme digestion analysis and DNA sequencing. The recombinant adenoviral vector pAdxsi - GFP - SLC7A8 was transiently transfected into NRK - 52E cells and the transfection efficiency was detected by flow cytometry. The level of expression of SLC7A8 mRNA was identified by reverse transcription - polymerase chain reaction ( RT - PCR ). Results The recombinant adenoviral vector was constructed successfully. The transfection efficiency was 94. 1% detected by flow cytometry. Over - expression of SLC7A8 mRNA was observed in NRK - 52E cells transfected with SLC7A8 gene. Conclusion The recombinant adenoviral vector pAdxsi - GFP - SLC7A8 was successfully constructed and over - expression of SLC7A8 gene in NRK - 52E cells was obtained. The recombinant adenoviral vector will be used to investigate the biologic role of SLC7A8 in hypertension.