摘要
目的:利用RNA干扰技术,以大鼠aggrecanse-1,2基因为靶基因,设计aggrecanse-1,2基因特异性的小干扰RNA(si RNA),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:根据Tuschl原则设计并合成大鼠aggrecanse-1,2基因特异性的DNA寡核苷酸,连接到经BamHⅠ、EcoRⅠ双酶切线性化的pKCSHR-GFP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8对大鼠aggrecanse-1,2基因特异性DNA寡核苷酸序列退火后,分别克隆到线性化的pKCSHR-GFP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测序鉴定确定为设计的序列。结论:成功构建了8对大鼠aggrecanse-1,2基因特异性shRNA重组慢病毒表达载体,可进一步用于干扰aggrecanse-1,2基因的mRNA转录,从而为制备aggrecanse-1,2基因表达受抑制的大鼠软骨细胞奠定基础。
Objective To design the small interference RNA （siRNA） specific to rat aggrecanse-1,2 gene by RNA interfering technique, construct its recombinant lentiviral expression vectors, and identify these vectors by DNA sequencing. Methods According to Tuschl＇s principle, the siRNA was designed and converted into cDNA of shRNA （small hair pin RNA） of siRNA for aggrecanse-1,2 gene. The cDNA was synthesized and inserted into plasmid pKCSHR-GFP which was linearized by restriction endonucleases BamH I and EcoR I . The recombinant plasmid was transformed into competent E. coll. DH5α cells. The positive recombinant colony was selected by ampicillin medium agar and identified by DNA sequencing. Results Eight recombinant lentiviral plasmids of siRNA specific to aggrecanse-l,2 gene were constructed successfully. Their DNA sequence analysis completely coincided with their designed sequences. Conclusion Lentiviral vector-based siRNA expression plasmids against aggrecanse-1,2 gene have been successfully constructed and identified. They will be further used for interfering aggrecanse-1,2 mRNA transcription and lay the foundation for aggrecanse-1,2 gene modified rat chondrocytes.
出处
《中国美容医学》
CAS
2007年第12期1679-1682,共4页
Chinese Journal of Aesthetic Medicine
基金
2005年国家自然科学基金资助项目(30572052)
2007年陕西省国际合作项目(2007KW-07)
