CD147 siRNA表达载体的构建与鉴定

Construction and Identification of CD147 siRNA Expression Vector

目的构建CD147siRNA表达载体,分析CD147在皮肤肿瘤浸润和转移中的作用机制。方法根据基因库上的CD147cDNA序列,设计并合成两端含有酶切位点的64个碱基的寡核苷酸链。寡核苷酸链退火后用T4DNA连接酶连接到线性化的pSUPER质粒中,并对重组质粒(命名为pSUPER/CD147siRNA)进行酶切、PCR及测序鉴定。结果PCR扩增片段与预期结果相符,双酶切证实CD147siRNA表达载体克隆构建成功,插入片段测序结果与合成的siRNA结果一致。结论成功构建CD147siRNA表达载体,CD147可为治疗肿瘤开辟新途径。

Objective To construct CD147 siRNA expression vector in o rder to analyze the role of CD147 in the invasion and metastasis of tumors deriv ed from skin.Methods According to CD147 cDNA sequence in the Genebank,a pair of 64-nt oligonucleotides,each containing the sites of restriction endonucleas e at both ends,were designed and synthesized.Oligonucleotides were annealed an d ligated with linearized pSUPER by T4DNA ligase.The recombinants (named pSUPER /CD147 siRNA ) were finally sequenced and identified by PCR and restriction end onuclease digestion.Results CD147 siRNA expression vector was successfully co nstructed and identified by double endonuclease digestion.Sequence analysis of inserted fragment revealed the same sequence as synthesized siRNA oligonucleotid es.Conclusions CD147 siRNA expression vector has been successfully constructed,which paves the way for studying the role of CD147 in the invasion and metasta sis of tumor cells derived from skin as well as in tumor therapy.