Separation And Purification Of A Novel Dna Enzymes In Nature And Immune Function

Earthworm nucleases are found during the process of extract anti-antiviral substances from earthworm.From what we have studied we can conclude that these group of nucleases have promising future to be anti-microbial and anti-tumor traditional medicines.These will help people to get a new idea in the probing new medicine of anti-virus.We have purified three kinds of DNases,PMF results have showed they are novel proteins.Earthworm low molecular weight deoxyribonuclease(ELMWD) is a kind of novel DNase that we have found.From the best of we have known,ELMWD is the first time to be studied.Our study will focus on the purify technique,Enzymatic mechanism,immune function of ELMWD.The methods and the results are briefly stated as followings:Methods and Results:1.Study on optimization deoxyribonuclease purify technology and purification Earthworm low molecular weight deoxyribonuclease(ELMWD)1.1 The study on expansion a kind of DNase activity measurementIn this chaptor,we introduced a new-type of activity measurement which have high resolution and can monitor the change of deoxyribonucleic number.DNaseI has been used to test the process.We have studied on how to eliminate the SDS impact on the activity of DNase.1.2 The optimization of deoxyribonuclease Chromatography sample preparation technologyThe nuclease Chromatography sample prepared in prophase experiment had much hybridprotein,mucoprotein,pigment which can affect the following chromatographic fractionation process,especially lead to the irreversible destroy on the column material.According to those information,three technical routes were proposed and verificated. Function gel and agar gel electrophoresis were used to monitor the numbers and activities change;Different methods were compared taking the activity to be reserved,the stickum and hybridprotein were removed as the main objective.The result showed that there were two kinds of DNases according to the electrophoresis mobility which had different tolerance to different elements.Two kinds of purify technical route were raised according to their characteristics.Mechnical homogenate,ammonium carbonate extract,heat denaturation(60℃,10min),pH 3.8 acetone fractional precipitation(or pH 4.6 acetone one time precipitation),acetone washing for three times methods were used to extract the slow electrophoresis mobility DNases.Mechnical homogenate,ammonium carbonate extract, ammonium sulfate fractional precipitation,acetone washing methods were used to extract the fast electrophoresis mobility DNase.1.3 The Study on purification of a kind of low molecular weight Lumbricus Bimastus DNase(ELMWD) through chromatography and electrophoresis methodsDEAE anion-exchange chromatography,hydrophobic chromatography were used to purify ELMWD from Lumbricus Bimastus.Function gel and agar gel electrophoresis were used to monitor the active peak and collect them.Based on the chromatography purifiction,2D-electrophoresis and 2D- electrophoresis function gel were combined used to purify ELMWD.2.The study on the biochemistry characteristics and enzyme kinetics of ELMWDIn this section,SDS-PAGE was used to determine the MWs of ELMWD as 23KDa. Two-dimensional electrophoresis and capillary electrophoresis were used to measure the isoelectric point as pH 4.5.Ultraviolet spectrophotometry,agar gel electrophoresis were used to study the temperature optimization and stabilities,pH optimization and stabilities of the ELMWD.MALDI-TOF was used to study the purity,MWs,PMF.The result was that the optimum pH was 5.2,the optimum temperature was 40℃.The ELMWD was sensitive to the temperature.Different metal ions and substrates were studied on the effect of the ELMWD reaction velocity.Enzymological studies showed Kms and Vmax of ELMWD was 0.040μg/μl, 0.059μg/μl·min-1;0.090μg/μl,0.059μg/μl·min-1 when taking calf thymus DNA and plasmid DNA as substrates respectively.Mg²⁺,Ca²⁺,Mn²⁺,EDTA can inhibit the activity of ELMWD, however,5mM K⁺ can stimulate the activity.The MWs according to MALDI-TOF was 20KDa. The result of PMF and the parameters of ELMWD suggested that the ELMWD was a novel DNase.3.The study on the relationship between the DNase from Lumbricus Bimastus and the innate immunity of Lumbricus BimastusAccording to the innate immunity angle of degrading inside or outside abandoned cells and microbes,we carried out the study of earthworm coelomic fluid activity extraction action on virus,bacterium,tumor cells in vitro;At the same time,The study of tissue distribution of DNase in Lumbricus Bimastus has made us better understanding the function of the DNase.3.1 The tissue distribution of DNase and ELMWD in Lumbricus BimastusIimmunoblot,immunohistochemistry,SREB,function gel were used to detect the tissue distribution of ELMWD and DNase.Immunohistochemistry results showed that the DNaseE principally exists in the circular muscle of its body wall and the mucous membrane of intestinal wall outside.Immunoblot and activity measurement results showed that DNase and ELMWD mainly located in the coelomic fluid,coelomic fluid cell,intestinal.The results suggested that DNases have participated in the innate immunity and digestion of microbies.3.2 The study on the anti-bacterium activity of coelomic fluid activity extraction(ECF) and ELMWDThe in vitro anti-bacterium experiment was conducted by the degradation of structure of bacterium layer by layer.The Fluospectrophotometry method was used to test the effect of the ECF on the integrity of the cytoplasmic and outer membrane of E.coli.Spectrophotometric was used to test the effect on peptidoglycan from Gram-positive bacteria.Agarose gel electrophoresis was utilized to study the nucleases activities on the genome and plasmid of bacteria.The result showed that the the ECF have the ability of non-specific degradation of the bacteria structure from outside to inside.Agar gel electrophoresis and biofilm formation method were used to test the bacteriostasis mechanism of DNase from earthworm.The result showed that the DNase can kill the bacterium through degrading the genetic materials combined with other immunological moleculars and can inhibit the forming of biofilm.3.3 The study on the anti-tumor activity ECF and ELMWDThe MTT and SRB method were used to test the the ECF from Lumbricus Bimastus on the inhibition activity of four kinds of tumor cells.Hochest fluorescein stain and single cell gel electrophoresis(SCGE) were used to test the genomic condition of tumor cells.The result showed that the ECF possess the notable activity of inhibition the tumor growth;the cytogene were degraded to fragments.Apotosis was tested to exist through Flow cytometry.MTT method was further used to test the antineoplasmic activity of ELMWD.The result showed that the ELMWD had the inhibition effect of invasion and metastasis but not the inhibition of growth of tumor cells.3.4 The study on the anti-virus activity of ECF and ELMWDWe have assayed directly the virucidal effect on three kinds of virus(including DNA and RNA virus) by cell culture,Inhibition of virus-induced cytopathogenic effect.The conclusion was that the ECF possessed non-specific ability of degrading virus,while ELMWD has not significantly anti-viral activity but can Collaborative the ECFChromatography analysis indicated that the anti-viral components are mainly located in three active peaks that possess proteases,nucleases and lysozymes,respectively.In addition,three active fractions mixture had stronger antiviral activity than three fractions alone but lower than the ECF.Gel electrophoresis showed that nucleases from Lumbricus Bimastus can degrade the genome of virus.The ECF displayed non-specific antiviral properties and maybe mediated by the cooperation of proteases,nucleases and lysozymes.Conclusions:1.A new activity measurement was introduced which has high resolution and can monitor the change of DNase numbers during the purification.This has set a foundation for later studies.2.Two sets of DNases crude extract preparation technical routes for two groups of different electrophoresis mobility DNases were established.3.A mature ELMWD purification technical route was set up4.A kind of novel high activity DNase was purified.It's molecular weight is 20759.417Da. It's affinity with liner calf thymus DNA is higher than with the plasmid DNA.The optimum pH was 5.2;The optimum temperature was 37℃.It's some enzymological kinetics characters and PMF result hint that this DNase is a kind of novel DNase different from others.5.Nuclease is one of the important elements of the innate immunity system.Nucleases play an important role in the degradation and inhibition microbes and abandoned cells in Lumbricus Bimastus independently or combined with other immune moleculars by degrading the genetic materials.It is significant to study and exploit the DNase from Lumbricus Bimastus.