Nucleoside（Acid） Anti-viral Drug Toxicity Evaluation Using Recombinant Human Mitochondrial DNA Polymerase γ

In recent years,nucleoside（acid）antiviral drugs have shown good prospects in the treatment of viral diseases,and their research,development and application have attracted much attention.Though nucleoside（acid）antiviral drugs are highly effective in the treatment of viral diseases,they can also have certain level of toxicity and side effects,especially on mitochondria.In this study,human mitochondrial DNA polγwas cloned and effectively expressed in yeast.Recombinant polγengineering enzyme amplification system was established and used to form a method of assessing mitochondrial toxicity of nucleoside（acid）analogues.The research methodology and results are as follows:P416-GPD-PLOG vector was firstly constructed.Human mitochondrial DNA polγwas expressed utilizing BY4741 yeast cells.The recombinant polγwas then obtained through purification.Recombinant polγengineering enzyme activity was detected and found slightly less active than natural mitochondrial DNA polγ.The engineering enzyme can be used for the screening and detection of nucleoside antiviral drugs.The recombinant polγengineering enzyme amplification reaction system in vitro was constructed using Real-time PCR.The optimum amplification condition of the recombinant polγengineering enzyme was found to be p H7.0.The optimum extension temperature was60℃and the concentration of Mg²⁺in the buffer was 2.0 mmol/L.Using Taq DNA polymerase and pfu DNA polymerase,the amplification of the product was detected by real-time PCR by adding azvudine triphosphate solution with different concentration gradient into the system.It was found that azvudine triphosphate could be recognized by Taq enzyme and pfu enzyme,and the amplification was inhibited with the increase of azvudine triphosphate concentration.The results suggest that the inhibition concentration was approximately 0.96 mmol/L～1.28 mmol/L under conventional amplification conditions.The DNA amplification curve catalyzed by recombinant polγengineering enzyme showed that under the influence of various concentrations of azvudine triphosphate,The ability of recombinant polγengineering enzyme to catalyze substrate amplification was inhibited,and the inhibition effect gradually increased with the increase of azvudine triphosphate concentration,and ultimately the substrate was completely inhibited and no longer amplified.It was found that the concentration of azvudine triphosphate with effective inhibition,under normal amplification conditions,was 12 mmol/L～16 mmol/L.When the concentration of azvudine triphosphate was set to 32 mmol/L,the substrate was completely inhibited.The amount of template also has significant impacts on the in vitro recognition of isonucleoside（acid）.When establishing the in vitro recognition system of isonucleoside（acid）,Both the isonucleoside concentration and the template amount need to be considered.If the template is excessive,the exponential period of the product amplification curve may be delayed.After the depletion of isonucleoside（acid）,the normal nucleotides in the system can still be amplified,thus affecting the analysis of the result and the recognition ability of the system.Based on the above research,we successfully established the in vitro molecular recognition system of nucleoside（acid）antiviral drugs using real time PCR technology and resolved the shortage issue of the mt DNA polγrequired for the in vitro toxicity detection and screening of nucleoside（acid）antiviral drugs,whereby laid the foundation for the in vitro toxicity detection and screening of nucleoside（acid）antiviral drugs.