Studies On The Production Technology Of DNA Vaccine

DNA vaccine and gene therapy as new ways shows a potential prospect. It is necessary for them to transfer external-originated gene into the host cell through vector to achieve therapeutic purpose. Common vectors include viral vector, in which there probably exists a hidden risk of security and plasmid DNA, which is inefficient in transfected cells. However, the requirement of plasmid DNA for gene therapy and DNA vaccination is increasing, so industrialized production of plasmid DNA is being paid more and more attention.The thesis is composed of five sections: I, determining the superior formula of cultural medium for the fermentation of plasmid DNA through the respond surface methodology; II, optimizing the parameter of the fermentation process through the experiments, adjusting the feed speed of making up to control DO and PH and lowering the growth speed ratio of the E. coli to improve the output of plasmid DNA; III, having studied systematically the correlative factors of large-scaled alkali lysis and their influence on the output and purity of plasmid DNA at different levels, designing an automatic and continual alkali lysis reactor on the base of traditional alkali lysis; IV, applying chromatography of different principles to purify plasmid DNA and try to find out the best elution condition of plasmid DNA; and according to this way, designing the large-scaled production technology of plasmid DNA; V, examining the quality of plasmid DNA in accordance with"Pre-clinical Studies Technical Guidance Principle according to Prevention with the DNA Vaccine"and establishing the quality system suiting the production technology.In response surface methodology, the screening of key factors of cultural medium is designed by the Plackett-Burman experiment which can use the least experiment numbers to screen fast and effectively factors with major influence based on the principle of incomplete equilibrium. The central composite design is a design of 5 levels, which can appraise the factors affecting the biological process and their correlation with limited experiment numbers and optimize the factors to achieve the optimum condition of biological process. According to the equation of plasmid production rate, analyzing by the response surface demonstrated by real value of parameter and carrying on the solution to the model equation, we can obtain that XB1B (glucose) =5.128 g/l. XB2B (peptone) =4.896 g/l, XB3B (sodium hydrogen phosphate) =6.085 g/l, XB4B (magnesium sulfate) =0.983g/l when the maximum value of the extreme value of the surface, i.e. response value (consistency of plasmid expression) is 75.28.Determining the fermentation condition through experiments: on the early stage of fermentation, DO is in 30-50% through the control of stirring number, airing quantity and pressure in a can; on the stage of feed, DO is in 30-50%, the PH value is in 7.0-7.2 through the affection of feeding speed so that the bacterium ratio growth speed is controlled at 0.2 or so. The field of plasmid DNA is 186.3ug/ml and the time of the fermentation is controlled in 20-22 hours. the filed of plasmid DNA and bacterium quantity are lower if the time is short; the field of plasmid DNA shows the tendency of dropping if the time is long; the fluid can run away if the time is too long.In the final process of plasmid production, the key step is E.coli lysis. In this process, the components in the bacteria (plasmid DNA, RNA, host DNA, the RNA endotoxin and protein) are all released. The release and retrieval of plasmid DNA will influence the result of the whole process. Because plasmid DNA is sensitive to shearing force, so the ultrasonic wave, the high-pressured physics method cannot be used to lysaze bacterium. Chemistry lysis is a better choice for industrialized production of plasmid DNA. First, comparing the alkali lysais and boiling lysis. The experiment proved that the field of the plasmid DNA is 77ug/ml in the alkali lysate, higher than 49ug/ml in the boiling. For E.coli DH5αstrain containing plasmid D-GPEi, the alkali lysis is quite suitable. Comparing the influence of different suspending volume, different lysis time, different proportion alkali lysis solution on the yield and purity of plasmid DNA, we draw the conclusion: when the proportion between wet weigh with the suspending solution is 1g/5 - 10ml; the lysis time is between 180 - 300s and the ratio between the suspending liquid, alkali lysis fluid and neutralization volume is 1: 1: 1, the yield and purity of plasmid DNA is the highest. Calcium chloride solution being joined into the lysis solution to cause high-molecule RNA, the protein, host DNA to condense and precipitate, and the solution viscosity to reduce, the difference between the lower solution and the upper suspending solution is more obvious, which is good for next step filtering. Contrasting the influence of calcium chloride in different concentration on the yield of the plasmid and the protein, the experiment proved that 0.5-1M/LCaClB2B can largely reduce the content of impure protein.The alkali lysis is widely used in the laboratory, but cannot be taken as a method in industrialized production. For example, when massive bacteria lysate, joining the alkali lysis solution will lead to viscosity phenomenon, which counteracts further response to cause the unevenness of the solution and the falling of lysis rate and the recover rate. The large-scaled alkali lysis reactor which we have established on the base of alkali lysis turns manual, interrupted alkali lysis operation to an airtight, continual industrialized production process. The bottleneck of the production has been resolved by 20-micron filters to clarify the lysis fluid instead of the centrifugation. It has been found that decontamination ability of the carbon filter is slightly stronger than the polypropylene filter. Then through ultra filters concentrates molecular RNA is further removed, and the conductivity of the lysis is adjusted through reducing volume and changing buffer solution, suiting for chromatography.Using the chromatography of different principles such as ionic exchange chromatography, the size chromatography, hydrophobic interaction chromatography etc. to purify plasmid DNA and try to find out the best elution condition of chromatography medium of every principle. According to the decontamination ability, the recovering rate of plasmid DNA, and the influence on the plasmid DNA configuration of each method, we obtain optimized technology of producing plasmid DNA. The purification order is: ionic exchange chromatography DEAE Sepharose FF (mainly removing RNA, protein); hydrophobic interaction chromatography Phenyl Sepharose 6 FF (mainly removing endotoxin, single-chain DNA); the size chromatography Sepharose 4 FF (mainly removing RNA, replace buffer solution); finally ultra filters for concentration to achieve medicinal dosage.Establishment the system of quality examination of the DNA vaccine according to "Pre-clinical Studies Technical guidance Principle according to Prevention with the DNA Vaccine", including: concentration determination of Plasmid DNA; restrictive enzyme qualification test of Plasmid DNA; protein residual determination; bacterium endotoxin determination; E .coli gene DNA determination; aseptic experimental unusual toxicity test; in vitro expression experiment; cellular immunity experiment and body fluid immunity experiment. The experiment shows that applying the optimized large-scale alkali lysis combining ionic exchange chromatography, hydrophobic interaction chromatography and size chromatography, this purification technology is able to produce plasmid DNA whose quality has achieved clinical standard.This purification process avoids using the enzyme originated from the animal (RNA enzyme), organic solvent (phenol; ethyl alcohol), toxic reagent (cesium chloride). The technology is simple and the production rate is high. And the technology is suited to the large-scaled industrialized production and of important practical significance for the industrialization production of the DNA vaccine...