Construction And Screening Of The Attenuated Trans-kingdom RNAi Bacteria

Trans-kingdom RNAi technology is an new gene-silencing technology crossing prokaryotic and eukaryotic kingdoms,which was first put forward by Shuanglin Xiang and his team.This technology constructed a trans-kingdom RNAi plasmid(TRIP)which contains gene inv,gene hly and gene fragments of transcribing shRNA.The invasion proteins,expressed by gene inv,are able to assist bacteria go across the cell membranes and enter into Eukaryotic cells.The listeriolysin O(LLO)proteins,through the expression of gene hly,help the bacteria flee from the hunting of phagosome inside the Eukaryotic cells.The Escherichia coli are called trans-kingdom RNAi strains when finishing the transformation of TRIP.The trans-kingdom RNAi strains can successfully break into eukaryotic cells and run away from phagosome's chasing,and then release transcribed shRNA interference fragments in the cytoplasmic,thus lead to the genes silence of eukaryotic cells.However,LLO expressed by gene hly is a strong virulence factor,on one hand,it can help bacteria flee from phagosome's tracking,on another hand,it does harm to the host cells.So,we aims to construct several point mutations on the protein LLO,and select attenuation strains from this mutant trans-kingdom RNAi strains.By overlapping PCR,we constructed 7 kinds of point mutant plasmids,namely K50A?R149A?D157A?K220A?R378A?Y427A?N432A.There were totally 9 types of such plasmids concluding ?PEST and G486D;the two were kept in the lab.We got 9 types of mutant trans-kingdom RNAi strains by transcribing these nine mutant plasmids into the E.coli BL21(DE3).In this experiment,we did PCR and found the nine strains contain gene inv and hly,which proved that these stains were the right and positive mutant trans-kingdom RNAi strains we needed.After that,we did Western blotting and cell infection tests,results of the two tests illustrated that the nine mutant trans-kingdom RNAi strains could still express LLO and be able to knock into eukaryotic cells.As we know that LLO do have hemolysis function,we did blood plate test,in vitro hemolysis test and MTT test so as to filter out mutant trans-kingdom RNAi strains with attenuation ability.The blood plate test was the first stage of screening,the results showed that nine types of strains had hemolytic ability,just like positive strains of TRIP,but the difference between their P hemolytic zones was not obvious.So,we had to do in vitro hemolysis test on these nine types of strains on the next stage.By vitro hemolysis test,we filtered out four types of mutant trans-kingdom RNAi strains with attenuation effect,the hemolytic abilities of the four strains were lower than that of positive strains of TRIP and all the four p values were lower than 0.05.The last step was MTT test,we did statistical analysis on the results,then we found that D157A,K220A,R378A,G486D still had attenuation effects.Besides,the p values for R378A?G486D were lower than 0.01,based on which we made reasonable speculation that stains R378A and G486D had the best attenuation effects.The mutant trans-kingdom RNAi strains will perform the functions of RNAi better after attenuation,and it will also promote the researches and utilizations of engineering bacteria.