水通道蛋白4基因克隆及其载体的构建

AQP4 Gene Cloning and Construction in Expression Vector

目的 克隆大鼠水通道蛋白 4 (AQP4 )基因并构建其载体为进一步研究提供理论基础和研究工具。方法 提取正常健康SD大鼠小脑皮质总RNA ,采用随机引物法进行逆转录构建大鼠小脑cDNA文库 ,应用自行设计的AQP4特异性引物 ,进行AQP4特异性扩增。利用设计的酶切位点进行相应的内切酶酶切后插入 pcDNA3.1Zeo( + )质粒中。结果 引物扩增出长度为 993bp的片段 ,为AQP4序列的全段 ,并正确插入到 pcDNA3.1Zeo( + )质粒中。上海生工生物工程技术公司片段测序结果与U14 0 0 7报道的序列相同。结论 正确克隆了大鼠AQP4基因并成功构建了其载体 。

Objective To provide AQP4 gene cloning and construction in the expression vector as a research tool. Methods Total RNA was isolated from rat cerebellar cortex, random primer was used for cDNA synthesis, and specific primer was used for PCR to amplify AQP4. PCR product amplified was digested with EcoR I and Not I. Then after complete digestion the DNA was inserted into pcDNA3.1/Zeo(+). Results The PCR product amplified by the pair of primer was 993bp, which is in AQP4 complete sequence (Genebank: U14007). The fragment was cloned into the pcDNA3.1 Zeo (+). It was sequenced at Sanggong, shanghai. The sequencing result was the same as reported. Conclusion AQP4 gene is cloned and constructed into the expression vector successfully, which can be used for further study.