大鼠尿素转运蛋白B基因克隆及真核表达载体的构建

Cloning of rat urea transporter B gene and construction of its eukaryotic expression vector

目的克隆sD大鼠尿素转运蛋白B（ureatransporterB，UT-B）的CDS全长基因并构建其真核表达载体。方法从SD大鼠肾髓质中提取总RNA并经逆转录反应获取总cDNA，以cDNA为模板，PCR扩增UT-B基因的CDS区全长片段，PCR产物加＂A＂尾后克隆至pMD-18T克隆载体，双酶切后获取UT-B片段，T4DNA连接酶将UT-B片段与KpnI和BamHI双酶切后的真核表达载体pcDNA3-1（-）连接，构建大鼠UT-B真核表达载体pcDNA3．1（-）-UT-B。结果成功克隆了sD大鼠UT-B基因，构建的pcDNA3．1（-）-UT-B载体经PCR扩增、KpnI和BamHI双酶切鉴定及测序验证，表达载体包含UT-B基因，且UT-B碱基序列与GeneBank中收录的大鼠UT-B序列-致。结论成功克隆了该基因并构建了其真核表达载体pcDNA3．1（-）-UT-B，为进-步进行UT-B基因转染治疗尿毒症的研究奠定了基础。

Objective To clone rat urea transporter B （UT - B） gene and construct its eukaryotic expression vector. Methods Total RNA was extracted from SD rat renal medulla and total cDNA was obtained by reversed transcription. The full length fragment of UT - B CDS part was amplified by PCR. The production of PCR was added an ＂A＂ tail and then inserted into pMD - 18T vector. The pMD - 18T - UT - B and pcDNA3.1 （ - ） vector were digested with restriction endonuclease BamH I and Kpn I, then T4 DNA ligase was used to ligate UT - B gene fragment and digested pcDNA3.1 （ - ） vector to construct the pcDNA3.1 （ - ） - UT - B eukaryotic expression vector. Results PCR assay showed that a DNA fragment of 1179 bp which was in accordance with the target fragment could be amplified. BamH I and Kpn I restriction endonucleases digestion confirmed the target DNA fragment had been inserted into the pcDNA3.1 （ - ）eukaryotic expression vector. The sequencing result verified the sequence of target DNA fragment was consistent with that included in Gene bank. Conclusion The full length of rat UT - B gene has been cloned and the eukaryotic expression vector has been constructed successfully, which lay a found for further study for uremia treatment by UT - B gene transfection.