Construction And Activity Analyzing Of M1GS Ribozymes That Targeting To The PB2 Gene Of Avian Influenza Virus

Aiming at the key replicase gene PB2 of the avian influenza virus,eight M1GS ribozymes were designed and constructed,which were T₄₃₆?T₉₃₄?T₁₁₀₅?T₁₅₂₃?T₁₇₇₁?C₃₄₁?C₁₅₃₁ and C₁₇₆₀.The cleavage activities of these M1GS ribozymes to the substrate RNA were examined by in vitro cleavage assay,the results showed that ribozyme T₄₃₆,T₁₁₀₅,T₁₅₂₃,C₁₅₃₁ and C₁₇₆₀ could specifically cleave the substrate RNA,and ribozyme C₃₄₁ was found to have strong non-specific cleavage activity,but among them the ribozyme T₉₃₄ and T₁₇₇₁ had no obvious cutting activity.By analyzing the electrophoretogram of the in vitro cleavage products and the relative activity of each ribozyme,three high active ribozymes,i.e.M1GS-T₁₁₀₅,M1GS-T₁₅₂₃ and M1GS-C₁₇₆₀ were screened.Accordingly,three eukaryotic M1GS expression plasmids(M1GS/T₁₁₀₅-PLXSN,M1GS/T₁₅₂₃-PLXSN,M1GS/C₁₇₆₀-PLXSN)and the green fluorescent protein expression plasmid?PB2-EGFP?were furtherly constructed.The three M1GS-PLXSN plasmids were respectively co-transfected with the plasmid PB2-EGFP into the MDCK cell.Results of western blotting showed that all the three M1GS ribozymes could effectively inhibit the expression of virus protein;The fluorescence scanning results show that M1GS-T₁₁₀₅,M1GS-T₁₅₂₃ and M1GS-C₁₇₆₀could all significantly decrease the fluorescence intensity,the inhibition rates were 77.53±1.98%,58.84±0.86%and 80.37±1.46%,respectively.Our study provided potential materials for subsequent experimental research,such as the antiviral activity of M1GS ribozyme in intracellular and even animal model,and layed a foundation for researching and developing a new kind of anti-AIV nucleic acid drugs.Moreover,we put forward some views about M1GS design.These datas were undoubtedly useful for the M1GS ribozyme-relevant research.