Construction and sequencing of recombinant lentiviral plasmid affecting human CCL20 gene by RNA interfering / 第三军医大学学报

ABSTRACT

Objective To design the small interference RNA (siRNA) specific to human CCL20 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 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 SpeⅠ and SalⅠ. The recombinant plasmid was transformed into competent E. coli. DH5? 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 interfering CCL20 mRNA transcription and lay the foundation for CCL20 gene modified human keratinocyte stem cells.