The Construction And Immunoreactivity Of DNA Vaccine Encoding BvrR Against Brucella

Objective:Brucella is the etiological agent of brucellosis which are facultative intracellular that causes abortion in domestic animals（cattle,sheep,and goats,suis）,and a febrile illness“undulant fever”in humans.Division of the genus Brucella into six classical Brucella species these species are Brucella melitensis,Brucella abortus,Brucella suis,Brucella ovis,Brucella canis,and Brucella neotomae.Now brucellosis is one of the most important zoonotic diseases in worldwide.Brucellosis is becoming one of the most serious public hygiene problem and is widely spread in north China such as Inner Mongolia,Shanxi,Jilin,Heilongjiang,Hebei,Ningxia and other provinces.At present,the prevention of Brucellosis for animals varies,according to the actual situation of districts,mainly is to use vaccine or quarantine culled method.So far,several live attenuated vaccines of Brucella are applied to production,B.abortus strain19（S19）vaccine,B.melitensis Rev.1（Rev.1）vaccine,B.abortus strain 45/20 vaccine and Rough strainB.abortus 51（RB51）vaccine which are mainy used in foreign countries.B.suis strain 2（S2）vaccine is widely used in our country.Though the live attenuated vaccines play a positive role in the preventing and controlling brucellosis,they are not safe for human,somes are considered too virulent.Depending on the dose administered during pregnancy,abortions will occur with variable frequency.For humans the Brucella attenuated vaccines have the possibility of potential infection.The live attenuated vaccines in aspects of simmune effect and the safety still exist some deficiencies.It is necessary to develop new vaccines of Brucella.Brucella as a parasitic intracellular pathogeny which can cause infection,live attenuated vaccines can stimulate strong CMI responses,they can be very effective vaccines against intracellular bacteria.The DNA vaccines seem to offer the best approach to activate cellular immune response.Furthermore,DNA vaccine shows many advantages over the traditional vaccines such as no risk of infection,induce a long-lived immune response,better stability than live attenuated vaccines,easy preparation,and low cost.Now there are reports about the DNA vaccines in preventing variety of virus,fungi and parasitic infection among people and animals.In the reserches of Brucellosis DNA vaccine,there are both outer-membrane protein DNA vaccine and intracellular protein DNA vaccine,the intracellular protein of Brucella as ribosomal L7/L12,Cu-Zn superoxide dismutase（SOD）lumazine synthase gene,have been demonstrated to induce significant levels of protection.This indicates that the Brucellosis intracellular protein can be a candidate gene as DNA vaccines.The BvrR/BvrS system is highly homologous to some two component systems of cell associated.The two-component regulatory system BvrR/BvrS has been conclusively implicated in Brucella virulence.According to reports,BvrR/BvrS avirulent mutants show reduced invasiveness in cells,and unable to inhibit lysosome fusion and to replicate intracellularly.Disfunction of BvrR/bvrS diminishes the characteristic resistance of Brucella to bactericidal polycations and increases its permeability to surfactants.As relatively conservative proteins which are not liable to vary,different species of Brucella BvrR/BvrS are highly homologous.If the regulatory protein can be used as a specific immunogenicity of DNA vaccine,then a kind of vaccine can prevent a variety of Brucella infections,it will be great significance in the field of specific preventing Brucellosis.So,in this study,we select BvrR gene in the BvrR/BvrS system,inserting eukaryotic expressing vector pcDNA3.1 to construct a new DNA vaccine,pcDNA-BvrR,and transfer it into Vero cells and then analyze the transient expression within the cell.Through the recombinant eukaryotic expression plasmid of BvrR gene（pcDNA-BvrR）immune the BALB/c mice research the immune effect of DNA vaccine.These reserches are done to explore the DNA vaccine immune mechanism and protective effect providing reference in further.We utilize the highly homologous in BvrR gene among different Brucellosis to produce a DNA vaccine in preventing the infection of different spices of Brucella,provide new ideas of treatment and prevention for Brucellosis.Methods:1.PCR amplification of BvrR gene.Obtaining PCR templates,use primer P1,P2 for PCR.After reaction to take 10μL reaction liquid for agarose gel electrophoresis,observaed electrophoresis results.2.The construction of prokaryotic express vector pET28a-BvrR.BvrR gene connected vector pET-28.Connection product transform competent cells,extract pET28a-BvrR plasmid.3.The express production of prokaryotic expression vector pET28a-BvrR analyse by western blotting.After transfer the membranes were blocked with 5%skimmed milk for 1h,respectively incubated with mouse anti-His and followed by corresponding second antibody,show color by DAB.4.The construction of eukaryotic express vector pcDNA-BvrR.BvrR genetic connected vector pcDNA.Connection product transform competent cells,extract pcDNA-BvrR plasmid.5.The express production of eukaryotic express vector pcDNA-BvrR analyse by RT-PCR and western blotting.Simply P total RNA extraction kit extracted total RNA,RT-PCR adopt two footwork kit（TaKaRa）according to manufacturer,reverse transcription by primer P2 for specific primer.After reaction to take 10μL reaction liquid for agarose gel electrophoresis,observation electrophoresis results.Preparation gel undertake SDS-PAGE polyacrylamide gel electrophoresis,according to gel area 1-2 mA/cm² for transfer,after transfer the membranes were blocked with 5%skimmed milk for 1h,respectively incubated with mouse anti-BvrR IgG and followed by corresponding second antibody,show color by DAB.6.pcDNA-BvrR DNA vaccine immune BALB/c mice.pcDNA-BvrR immune group,pcDNA control group respectively injected 1μg/μL plasmid pcDNA-BvrR and plasmid pcDNA sum to 100μg on tibialis anterior.PBS control group injected PBS 100μL on tibialis anterior.Interval two weeks injected again,total immune three times.7.Detected mice Specific antibodies in serum and Cytokines by ELISA.Detected mice the Specific antibodies and isotype IgG1 and IgG2a level in serum and the IFN-?、IL-4 level in serum by ELISA to confirm the immunity effect of pcDNA-BvrR DNA vaccine.8.The MTT method detected mouse spleen lymphocyte proliferation.9.Flow cytometric analysis T cells subtypes of mice（CD4⁺,CD8⁺）.10.Confirm protection ratio by protection experiments.The mice of pcDNA-BvrR immune group and pcDNA control group were challenged with a B.abortus S19（2×10⁶CFU/μL）on tibialis anterior after7 days third-immune,after 14 days the spleens were removed aseptically,homogenized with 2ml of NS,plated in selective medium of Brucella.plates were incubated at 37℃with 5%CO₂,counted and expressed as log CFU per spleen.Results:1.Using polymerase chain reaction（PCR）amplify the BvrR gene.2.Constructing the prokaryotic express vector pET28a-BvrR.The express production of prokaryotic expression vector pET28a-BvrR analyse by SDS-PAGE and western blotting was fusion protein.There existing immunoreactivity between the purified BvrR protein and polyclonal antibody of Brucella.3.Constructing the eukaryotic expression vector pcDNA-BvrR.The express production of eukaryotic express vector pcDNA-BvrR analyse by RT-PCR and western blotting.4.pcDNA-BvrR DNA vaccine stimulate mice produce specific antibodies IgG.5.pcDNA-BvrR nucleic acid vaccine enhance secretion of IFN-?,t-cells sub-types（CD4⁺,CD8⁺）,stimulate lymphocyte proliferation.6.pcDNA-BvrR DNA vaccine induct a typical Th1-dominated immune response.Conclusions:1.There existing immunoreactivity between the purified BvrR protein of prokaryotic expression and polyclonal antibody of Brucella.2.The pcDNA-BvrR DNA vaccine stimulate mice produce specific antibodies IgG,induct a typical Th1-dominated immune respons.3.The pcDNA-BvrR DNA vaccine resulted in a higher degree of protection.