The Expression And Purification Of Mitochondria Polymerase γ Gene

The medicines of Nuclei(nucleic acid) analogs could affect significantly on treating virus infection among the medicines of anti-virus, in which one of mechanism was to compete with substrate of DNA polymerase to suppress DNA polymerase, and further to prevent DNA replicating and control the virus load in the infectors. The medicines of Nuclei(nucleic acid) analogs, such as Lamivudine, adefovir dipivoxil, ETV, Tibifuding and tenofovir were allowed to treat chronic hepatitis B(CHB),and the former 4 kind of medicines can be got on the marker in China, and tenofovir will also be put into the clinical trial.The medicines of Nuclei(nucleic acid) analogs suppress the activity of DNA polymerase of virus,and meanwhile, influence the replication of DNA in human beings. Because of the chronicity of treatment of hepatitis B, the toxic effect of these kinds of medicine on the DNA polymerase γ in the cells of human beings will gradually cumulate,and then harm to mitochondria. As a result of these, the toxic evaluation of the medicines should be done in the research of newly-developed isonucleotide and its analogs, in order to the selecting and finding of medicines which can be widely used in clinic, characterized with low toxicity and high coefficient of anti-virus, then these works will play an important part in the control and cure of infectant disease of virus.In this study, we obtain two c DNA fragments of mitochondrial DNA polymerase γ from a human cell line(Hela Cell) by RT-PCR. The p MD18-T vector was applied to clone and characterize the two fragments genes of human mitochondrial DNA polymerase γ in E. coli JM109 and did the phylogenetic analysis. The p ET28-p138 expression vector was constructed by using PCR specific truncated mutant gene and then transformed into E. coli EL21(DE3) to induce expression and inclusion body refolding of recombinant protein. The results showed that we obtained the active enzyme protein successfully.The result indicated that there were more varities on gene of DNA polymerase in mammals and differences varied more among them, so it was necessary to colne human beings gene when researching DNA polymerase of human mitochondria. we established procaryotic expression system of polymerase γ of mitochondria, providing the basic reearch for getting polγ polymerase in mass, and also providing the novel method and technology for engeering enzymes with different functions..