Abstract
Objective To construct a recombinant lentivirus plasmid of RNA interference (RNAi)of RelB gene.Methods According to the GenBank information of RelB,5 complementary DNA containing both sense and antisense Oligo DNA of the targeting sequences were designed,synthesized,and cloned into PlentiLox 3.7 which contained U6 promoter and green fluorescent protein(GFP).The positive clones were confirmed by PCR,sequencing,and authentication with KspA Ⅰand Hind Ⅲ.RM-1 cells were transfected and detected by Western-blot.293T cells were cotransfected with lentiviral vector and virus packaging plasmids.All virus stocks were produced by calcium phosphate mediated transfection.Results PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of RelB was constructed successfully.The most effective siRNA sequence was 5'-TTGGAAATCATCGACGAAT-3'.The titer of concentrat ed virus was 8×1010 pfu/L.Conclusion The lentivirus RNAi vector of RelB was constructed successfully.The lentivirus RNAi vector of RelB significantly inhibited the RelB expression. Key words: siRNA; Lentiviral vector; Construction; Identification
Published Version
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