Enhancing The Thermostability Of Leukemia Drug L-asparaginase II & Construction, Purification And Evaluation Of DNA Vaccine For Minimal Residual Leukemia

In the treatments of acute lymphoblastic leukaemia, there are two problems puzzling people all alone: One is the side-effects caused by chemotherapy; and another is the minimal residual leukaemia which leads to the relapse of cured patient. The present study has dong some researches to try to solve these problems.L-Asparaginase II of E. coli is a kind of effective drug in the treatment of acute lymphoblastic leukaemia. However, during asparaginase therapy, repeated using of the drug is commonly needed because of the enzyme's relatively short half-life and instability in the processes of production and treatment. This leads to more serious toxic effects on patients. In order to stabilize the enzyme, a higher thermostable mutant L-asparaginase II was created in the present study by replacing Aspl78 with proline in a hydrogen-bonded turn (178-180DGR) which is contribute to the thermostability of the enzyme. The results displayed that values of K_m and K_cat for the mutant enzyme are not affected although the energy of activation is increased comparing to the wild-type enzyme. These data suggest that such alteration for L-asparaginase II enhances the thermostability of the enzyme without changing the enzyme's activity and thus the therapeutical use of L-asparaginase II might be benefit from these results.In present study, the immune-therapy method has been explored when it comes to the problem of minimal residual leukemia (MRL). Immunized with the DNA vaccine is a potential way to treat MRL for its better safety comparing to the tumor cells vaccines. In present study, a two-plasmid vaccine system which is used to express a tumor-special antigen (Survivin) has been constructed and immunized with mice. Furthermore, a barrette-like ssRNA, GM-CSF and a co-stimulating factor CD80 have also been co-expressed in the same system. The results displayed that strong T cell immune response and CTL activity were induced by the DNA vaccine. It demonstrated that DNA vaccine is a promising immune-therapy method to fight against MRL.Techniques for large-scale preparation of clinical-grade plasmid DNA serve as an important base for clinical trials and industrialization of DNA vaccines. In the present study, we compared the performances of size-exclusion chromatography for the purification of plasmid DNA when different concentrations (0.5 M, 1M, 2 M, respectively) of two types of salt (NaCl and (NH₄)₂SO₄) are present in running buffers. Our experiment results displayed that it is not only the resolution of RNA but also those of supercoiled plasmid DNA and host's genomic DNA were increased greatly in the presence of high concentration of water-structure salt. We deduce that two separation modes may be involved in the process: The supercoiled plasmid DNA is influenced mainly by compaction effect and eluted in the size-exclusion mode; whereas, RNA and genomic DNA are influenced mainly by hydrophobic effect due to their stretched and loose structures and eluted in the interaction mode. This method led to an improved efficiency of size-exclusion chromatography.