摘要
目的构建携带色氨酸羟化酶（TPH）-2基因的小分子干扰RNA表达载体。方法设计并合成shRNATPH-2对应的两条互补的寡核苷酸链，pU6-CMV—GFP质粒经AgeI和EcoRI双酶切与退火后的寡核苷酸连接，目的质粒转化感受态细胞，对长出的克隆应用菌落聚合酶链反应（PCR）鉴定，再对PCR鉴定阳性的克隆进行测序和比对分析。结果经PCR、酶切及测序证实，pU6一CMV—GFP—TPH-2shRNA表达载体片段大小为332bp，其中插入的片段序列和位点与预期完全一致。结论实验成功构建pU6-CMV—GFP—TPH2shRNA表达载体。
Objective To construct and identify recombinant TPH-2-targeted small interfering RNA （siRNA） expression vector. Methods Oligonucleotide containing the small hairpin of TPH-2 was designed and synthesized, which was inserted into the pU6-CMV-GFP plasmid double digested by Age I and EcoR I. The aim plasmids were transformed into competent cells E. coli DHSα. The grown colonies were identified by colony polymerase chain reaction （PCR） and then the positive colonies were sequenced and aligned. Results The result of PCR and gene sequencing confirmed that the pU6-CMV-GFP-TPH-2 shRNA expression vector with 332 bp. Conclusion The expertment had been constructed successfully.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2011年第11期1981-1982,共2页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（81071307）
关键词
色氨酸羟化酶
RNA干扰
表达载体
Tryptophan hydroxylase
RNA interfere
Expression vector
