Study Of Purification And Analysis Of Plasmid DNA D-GPEi

Gene therapy and DNA vaccine show future as a new generationtherapy .they need to introduce into human cells through the vector inorder to modify their genetic repertoire for therapeutic purposes.Generally speaking, vectors could be divided into viral vectors andnon-viral vectors, and using viral vectors tends to cause potentialsafety hazard. Plasmid-DNA-based delivery vectors are less effectiveat transfecting cells. Moreover uses in the gene therapy and DNAvaccine plasmid DNA demand increases, the industrializationpreparation for medicinal purposes level, the purification of plasmidDNA receives the attention gradually.The paper study bacterial lysis of the correlation factor as well asvarious factors of different level to the plasmid DNA field and purityinfluence;And applies the different principle chromatography to carryon the purification of plasmid DNA, tried to find out each kind ofprinciple chromatography elutes the condition best regarding plasmidDNA. Designs according to this suits in large scale production ofplasmid DNA, and according to 《Prevention Clinically Front toTechnical guidance Principle with the DNA Vaccine》 carry on thequality analyse to plasmid DNA.The first critical step in the downstream processing of plasmid DNAis cell lysis, In this step, all the intracellular components, includingplasmid DNA, RNA, gDNA , endotoxins and proteins, are released. Inthis step, All the intracellular components, including plasmid DNA,RNA, gDNA, endotoxins and proteins, are released. The shearsensitivity of plasmid and gDNA molecules, as well as the highviscosity of the lysates15, are of major concern during plasmid release.Because plasmid DNA is sensitive to the shearing force, cannotuse the, the high-pressured refining physics method lysis bacterium,chemistry lysis is good choice for the downstream processing ofplasmid DNA. First we withdraw plasmid DNA to the alkali lysis andboil the lysis to the plasmid DNA two methods to carry on thecomparison, the experiment proved in field of plasmid DNA aspect,alkali lysis withdraws the plasmid DNA is 77ug/ml, is higher than bmethod 49ug/ml. Regarding withdraws includes the material particleD-GPEi Ecoli DH5α strain to say that, the alkali lysis law is quitesuitable.In the fermentation process, obtains wet weight above 60g/L, thesuspend extremely is generally important regarding the plasmid DNAoutput, the great volume suspending liquid is advantageous to suspend ,but also as a result of oversized volume influence following operation,not only can cause the cost to enlarge, moreover lengthens theproduction cycle. Through the contrast different suspend volume to theplasmid DNA output influence, we obtains the conclusion works as themycelium wet weight and the suspending liquid proportion when1g/5-10ml, the plasmid DNA output basic has not come under theinfluence.In the alkali process, the lysis time influence plasmid DNA fieldand purity, the lysis time excessively is short, majority of materialparticles DNA cannot release from the cell, but if the lysis timeexcessively is long, also can cause the plasmid DNA denaturation forthe split-ring or the linear form. The experiment proved the lysis timebetween 180-300s, ultra spiral plasmid DNA and the impurity outputwhich produces is highest。In the alkali process, the pH controls plasmid DNA in 12-12.5 theoutput and the purity is advantageous. If lysis fluid pH excessively islow, host DNA cannot the complete denaturation, separate verydifficultly with plasmid DNA, the increase purifies the craft thecomplexity. On the other hand, if the lysis fluid pH value excessivelyis high, can cause the ultra spiral plasmid DNA denaturation for thesplit-ring and the linear form. Through the contrast differentproportion alkali lysis response fluid to the alkali lysis process in theplasmid DNA influence, we draws the conclusion: The myceliumsuspending liquid/alkali lysis fluid/center and the fluid volume ratio is1: 1: 1 plasmid DNA output is high.Joins the calcium chloride solution in alkaline lysates to causemacro-molecule RNA, protein, host DNA to condense, precipitation,the solution viscosity drop, the lower part solution level is moreobvious with the upside aerosol level dividing line, is advantageous innext step filters. Contrasts the different calcium chloride end density tothe plasmid DNA output and the mixed protein influence, theexperiment proved that, 0.5-1M/LCaCl2can reduce the mixed proteincontent largely.Chromatography elutes the condition best. Removes thecontamination,the field of plasmid DNA,to the plasmid DNAconformation influence based on each method, we gained theoptimized processing of plasmid DNA. The purification sequence is:DEAE Sepharose the FF(IEC), Phenyl Sepharose 6 FF (low sub)(HIC), Sepharose 4 FF(SEC), finally carry on ultra filter theconcentration to achieve the for medicinal dosage.Endotoxin removing is one of entire plasmid DNA purificationbottlenecks, the reason lies in the endotoxin and plasmid DNAcompares, the molecular weight and the electric-charge densityapproach very much. The endotoxin molecular weight from106-108,has the massive negative charges, this meant, the use at present thelarge-scale preparation for medicinal purposes plasmid DNA SEC andtIEC is very difficult for endotoxin to fall to the very low level.In order to solve this difficult problem, we introduced HIC toeliminate the endotoxin, obtained the tangible effect. Because plasmidDNA has the very strong hydrophile, after on the type did not unifywith hydrophile groups, forms the class to put on the peak. Butendotoxin have hydrophobe character, with hydrophobe groups, whenreduces gradually elutes concentration , is eluted . Thus achievedremoves the endotoxin the goal. Because HIC purification plasmidDNA belongs to the negative adsorption, endotoxin are adsorbed all onthe medium, therefore the earlier period needs through purificationmethods and so on high salt precipating ion exchange to reduce inlarge scale the sample the pollutant density, achieves the good effect.HIC obtains the sample includes the high salt, replaces the appropriatebuffer solution through SEC and further removes the contamination.Clinical Front Studies Technical guidance Principle according toPrevention with the DNA Vaccine, we established the DNA vaccinequality to examine the platform, including: The plasmid DNA densitydetermination, plasmid DNA restrictive inscribes the enzymequalification test, the mixed protein residual content determination, thebacterium endotoxin content determination, the E.coli genome DNAdetermination, the aseptic experiment. The experiment indicated, theuse optimizes the alkali lysis law union IEC, the sparse waterchromatographic analysis, the gelatin filter the chromatographicanalysis, this purification craft prepares the plasmid DNA quality hasachieved the clinical practice standard.This purification craft avoids using enzyme (RNaseA) originatingfrom animals, the organic solvent (the phenol;EthanolA.R), and thetoxic reagent (cesium chloride). This production craft is simple, theproduction rate is high, and a platform has also been established toexamine DNA vaccine quality. This craft is fit for mass production,which bears realistic meaning with regard to the industrial productionof DNA vaccine.