Study On Continuously Thermal Lysis Preparative Protocol Of DNA Vaccine Against Foot-and-mouth Disease Virus

At present, the direct application of genes as vaccines and for medical therapy shows great promise. Plasmid DNA has been widely used as a gene delivery vector in many cases. However, Plasmid-DNA-based delivery vectors are low effective in transfecting cells,with full treatments requiring milligram quantities of plasmid DNA. Therefore, large-scale plasmid manufacture processes must be developed as gene therapy and DNA vaccines move from the laboratory to clinical trials and market approval. Alkaline lysis is the traditional protocol for plasmid DNA preparation in laboratory, However,it encounters great disadvantage when the scale was enlarged Our studies concentrated on the protocol of large-scale preparation of FMDV DNA vaccine pcD3-VP1. Based on the previous studies about the protocol of plasmid preparation, we try to find a new one, which is effective and economical, can be used in large-scale production of FMDV vaccine. A protocol on large-scale preparation of plasmid with the ability of effectiveness and low cost was valuable in pharmacy industry. Host Escherichia coli take an important role in plasmid preparation, for the replication of plasmid taken place in cytoplasm. The metabolism and growth condition of host directly affect the yield of plasmid. Four host strains DH5α,JM109,TOP10 and XL1-blue were transfected with pcD3-VP1 respectively, then cultured in same conditions. Compared the plasmid contain rates of four strains after culture. The plasmids were extracted from four strains and selected by the standard of the yield. As a result, the strain TOP10 was the best one both in plasmid contain rates and plasmid yields. TOP10 strain was selected in later experiments. The primary goal of the fermentation is to maximize the amount of plasmid DNA that is made. The yield of plasmid was directly related to the amount and the ability containing plasmid of the host. Different ingredient of culture medium would affect the growth of host and the replication of plasmid DNA. Feeding strategy of nutrition is the key to a high-density fermentation. In our experiment, four different mediums were applied in fermentation, which had different C/N and beginning density of nutrition. Three nutrition-feeding modes were tried in the fermentation of TOP10 strain. The results of cell growth status and plasmid yield during fermentation indicated that an appropriate proportion of carbon and nitrogen and nutrition density during fermentation took a critical role in plasmid yield. Another result showed that the amounts of hosts and the yields of plasmid did not take a linear correlativity. This illuminated that the capability of containing plasmid copies in the host grown under different circumstance was dissimilar, or, hosts in different status affect the preparation of plasmid. In some experiments, using semi-synthesize medium and constant dissolve-oxygen feedback nutrition feeding mode, E.coli culture achieved a density of OD600 around 50 and plasmid yields was the highest. The critical step in the downstream processing of plasmid DNA is cell lysis and this step is the bottlenecks step in Large-scale preparation. Alkaline lysis is the traditional method in cell lysis, and which was wildly used in large-scale preparation of plasmid DNA now. But alkaline lysis has some huge obstacle when the scale was enlarged. The biggest one is that it will be difficult in very large mixing tanks to achieve a homogeneous concentration of the lysis chemicals (NaOH and SDS), since the solution viscosity rapidly increases and becomes non-Newtonian. Local regions of high (>12.5) pH can irreversibly denature the DNA plasmids. On the other hand, the stir must be soft for the plasmid DNA and genome DNA were very sensitive to the shear. If the genome DNA were broken into pieces by the shear, it is almost impossible to separate the plasmid and gDNA pieces in later process. And alkaline lysis also has disadvantages such as low yield rates and bad repeatability. In our experiments, a continuously thermal lysis was used to cell lysis. In this process, a constant pump was used to drive the cell flowing through a thermal exchange coil to finish lysis and release plasmid. Flowing through the thermal exchange coil, cells receive an identical thermal intensity in spite of the volume of the sample, which guarantees the continuity and homogeneity for the cell lysis. After optimization, the lysis temperature was down to 70℃and lysis time down to 20 sec. The continuouslythermal lysis has simple operation and short process time, and the instruments were very easy to enlarge to industry manufacture. Using this protocol, about 100mg plasmid DNA was prepared from per liter culture. The plasmid DNA taken by continuously thermal lysis has the same character as the plasmid taken by alkaline lysis. And both humour and cell response to FMDV can be induced in the mice immunized with the plasmid pcD3-VP1 Traditional methods of plasmid purification were used in our experiments to purify the plasmid DNA after the step of continuously thermal lysis, and it made whole process un-linearized. The next step of our work is the plasmid separation and purification after the step of continuously thermal lysis. The potential methods comprise tangential flow microfiltration and preparative electrophoresis. The continuously thermal lysis will offer an active effect in large-scale preparation of plasmid DNA.