The Construction Of HIV-targeted Trans-kingdom RNA Interfering Bacteria By Red/ET And Its RNAi Application

Transkingdom RNA interference is a novel approach for siRNA delivery,which employs the geneticly engineered bacteria to produce siRNA in targeted mammalian cell.The siRNA carrying bacteria can invade into target cell under oxygen-deficient environment or with the help of receptor on the target cell,after which the bacteria was cracked in the cytoplasm and release the siRNA to induce specific gene silence.The technique solved the problem of the stability,targeting and delivery efficiency of siRNA.For the application of this technique to treat HIV,transkingdom RNA interference bacteria for T-lymphocytes should be first constructed.To reach this goal,in this thesis,transkingdom RNA interference plasmid Trip was constructed.The construction was carried out in two aspects:first,in order to avoid the interference failure owing to the high mutation and variation of HIV,the attack range should be expanded besides the conservative area.By using the widely existing RNA enzyme ? in bacteria,which can be used to cut the long double-stranded siRNA into short strands to enhance gene silencing effeciency,the attack range can be expanded,and the interference will be achieved even with a few mutations.This is benifitial for the treatment of highly mutational HIV.We construct the TRIP plasmid in order that the T7 promoter expresses the long double-stranded RNA instead of short hairpin interfering RNA.The other aspect is to reduce its biological toxicity.Thus,low toixic transkingdom RNA interference plasmid was constructed.Then,Red/ET homologous recombination technique was utilized to transform the low toixic Trip plasmid into Escherichia coli BL21(DE3),which turned E.coli BL21(DE3)into a transkingdom RNA interference bacteria for HIV treatment.The constructed transkingdom RNA interference bacteria was then used to infect SW480 containing the key gene nef of HIV.Finally,the protein expression of the transkingdom RNA interference bacteria was detected by Western Blot.