人CCL20基因特异性shRNA重组慢病毒表达载体的构建及测序

Construction and sequencing of recombinant lentiviral plasmid affecting human CCL20 gene by RNA interfering

目的利用RNA干扰技术,以人CCL20基因为靶基因,设计CCL20基因特异性的小干扰RNA(siRNA),构建其短发夹RNA(shRNA)重组慢病毒表达载体,并进行测序鉴定。方法设计并合成人CCL20基因特异性的DNA寡核苷酸,连接到经SpeⅠ和SalⅠ双酶切线性化的pHSER dsRNA GFP SIN质粒上,转化大肠杆菌DH5α感受态细胞,筛选阳性菌落、扩增后提取质粒,进行DNA测序鉴定。结果将合成的两对人CCL20基因特异性DNA寡核苷酸序列退火后,分别克隆到线性化的pHSER dsRNA GFP SIN载体质粒上。在氨苄青霉素培养基条件下,筛选出相应的阳性菌落,经DNA测序鉴定确实为所需序列。结论构建成功了2对人CCL20基因特异性shRNA重组慢病毒表达载体,可进一步用于干扰CCL20基因的mRNA转录,从而为制备CCL20基因表达抑制型的人角朊干细胞奠定基础。

Objective To design the small interference RNA （siRNA） specific to human CCL20 gene by RNA interfering technique, construct its recombinant lentiviral expression vectors, and i DNA sequencing. Methods According to Tuschl＇s principle, the siRNA was designed dentify these vectors by and converted into cDNA of shRNA （ small hairpin RNA） of siRNA for CCL20 gene. The cDNA was synthesized and inserted into plasmid pHSER-dsRNA-GFP-SIN which was linearized by restriction endonucleases Spe Ⅰ and Sal Ⅰ. The recombinant plasmid was transformed into competent E. coli. DH5ot cells. The positive recombinant colony was selected by ampicillin medium agar and identified by DNA sequencing. Results Two recombinant lentiviral plasmids of siRNA specific to CCL20 gene were constructed successfully. Their DNA sequence analysis completely coincided with their designed sequences. Conclusion Lentiviral vector-based siRNA expression plasmids against CCL20 gene have been successfully constructed and identified. They will be further used for interfeting CCL20 mRNA transcription and lay the foundation for CCL20 gene modified human keratinocyte stem cells.