生长抑素受体基因特异性shRNA慢病毒表达质粒的构建

Construction and Identification of shRNA Lentiviral Expression Plasmids Targeting into SSTR5 Gene

目的:设计并合成生长抑素受体SSTR5基因siRNA序列,并构建其短发夹shRNA慢病毒表达质粒。方法:以小鼠SSTR5基因为靶序列,用在线软件分析、设计并合成其有效siRNA,退火形成双链DNA后,与经BamHⅠ和EcoRⅠ双酶切线性化慢病毒表达载体pSHR-Pμzro/GFP连接,产生pLV-shSSTR5重组慢病毒质粒,将重组质粒转化大肠杆菌DH 5α感受态细胞,PCR筛选阳性克隆,测序鉴定。结果:构建的重组表达质粒PCR产物为161bp,其中插入的SSTR5-siRNA片段为61bp,测序结果与参考序列完全一致。结论:成功构建了小鼠SSTR5基因特异性shRNA慢病毒表达质粒。

Objective： To design and construct siRNA lentiviral expression plasmid targeting mo use SSTR5 （somatostatin receptor） gene by RNA interfering technique. Methods： According to mouse SSTR5 gene deposited in Genbank, the oligo primers targeting mouse SSTR5 gene were designed and synthesized. The PCR product was cloned into linear pSHR-Pμro/GFP by BamH I and EcoR in order to construct a siRNA expression plasmid. Results： The PCR product of constructed plasmids was 161bp, in which, inserted SSTR5-siRNA flagment was 61bp, and DNA sequencing demonstrated that the inserted fragrnents were consistent with the reference sequences. Conclusion： The lentivirus RNAi vector of SSTR5 was constructed successfully, which provides a tool for further study in the effects of SSTR5 gene expression on mouse growth with RNAi technology.