Construction and identification of a short hairpin RNA expression vector targeting the Cbl-b gene

Objective To construct a eukaryotic expression plasmid vector encoding Cbl-b gene-specific short hairpin RNAs（shRNAs）, and to evaluate its interference effect, so as to lay a foundation for further study on the role of Cbl-b in the immunotherapy of malignant melanoma. Methods According to the sequence of Cbl-b cDNA, 4 pairs of shRNAs targeting the Cbl-b gene were designed and synthesized, and then inserted into the plasmid PGPU6/GFP/Neo to construct recombinant plasmids. After identification by DNA sequencing, the 4 shRNA expression vectors were co-transfected into 293T cells with the Cbl-b gene eukarytic expresson plasmid, respectively. The knockdown efficiency of these shRNA expression plasmids on Cbl-b expression was evaluated by real-time（RT）fluorescence-based quantitative PCR and Western blot at 48 hours aftert transfection. Results Sequencing analysis revealed that all the 4 pairs of shRNAs were successfully inserted into the eukarytic expression vector PGPU6/GFP/Neo. As RT-PCR and Western blot showed, all the 4 shRNA-expressing vectors could downregulate Cbl-b expession, and the NO.1 shRNA-expressing vector displayed the strongest interference effect（P < 0.05）. Conclusions A eukaryotic expression plasmid vector was successfully constructed for Cbl-b gene-specific shRNAs, and the most effective shRNA was selected in this study. Key words: Ubiquitin-protein ligases; RNA, small interfering; Melanoma; Genes, Cbl-b