The Cloning,Expression And Immune Protection Of Acinetobacter Baumannii A1S₁969α Outer Membrance Protein

Objective:In this study,we aimed to eveluate wether A1S₁969α protein couled serve as an Acinetobacter baumannii vaccine candidate antigen.Frist,cloneing,expression A1S₁969α recombinant protein through genetic engineering technology.Second using affinity chromatography to prepare high-purity A1S₁969α recombinant protein,then companying with aluminum hydroxide adjuvant to prepare A1S₁969α protein immunogen.Third,building a sepsis mouse model by infection ATCC 17978 strain through intraperitoneal injection.At last,to evaluate A1S₁969α recombinant protein whether can enhance mice to prevent the lethal infection by ATCC 17978 strain on sepsis mouse models,statistical and analysis the levels of serum inflammatory cytokine,microbial loading in viscera and Pathological changes in lungs in 12 hours and mice survival rate in 7 days after lethal infection,to evaluate the immune protective of A1S₁969 protein antigens.And do some preliminary research for the structure and function for A1S₁969 protein.Method:1.Analyzing A1S₁969 with bioinformatics software.Extracted the clinical Ab strainS genome,and detect A1S₁969 gene sequence in these clinical strains by PCR.2.ATCC 17978 Acinetobacter baumannii is a international standard strain which buy from United States,grow in LB Liquid culture for 12 hours,and Extract the whole genome as a template for acquiring A1S₁969α purpose gene by PCR.Using Bam HⅠand NotⅠendonuclease to digest A1S₁969α gene,then insert to pGex-6p-2 plasmid vector,finally,the plasmid recombinate into Ecoli XL1-Blue building PGEX-6 p-2-A1S₁969α/ XL1-Blue genetic recombinant strains.3.IPTG is Used to induct PGEX-6 p-2-A1S₁969α/ XL1-Blue strains to express A1S₁969α recombinant protein.To obtain A1S₁969 recombinant protein.First,extracte GST-A1S₁969α fusion protein by affinity chromatography.Then PreScission protease is used to enzyme digestion GST label.Finally,through G25 column and QHP column replacing buffer and removing endotoxin to obtain pure A1S₁969α recombinant proteins.HPLC,SDS-PAGE,BCA and used to detect the purity and concentration of the protein,also through Mass spectrum Capillary electrophoresis to detect N-terminal amino acid residue and molecular weight of A1S₁969α recombinant protein4.ATCC 17978 standard strains grow in LB Liquid culture under 37℃ and draw the growth curve within 12 hours.Collect the bacteria which in logarithmic phase and infection BALB/c mice With different amount of bacteria,through Statistics of the different groups of BALB/c mice survival rate to find the LD50 and LD80.Uuing LD50 bacteria dose to infect BALB/c mice,and detecting the bacterias colonize in blood,liver,kidney,spleen and lung at different times.5.In this sduty,through detecting antibody level of mice in 60 days after immune with A1S₁969α recombinant protein,also detect the antibody subtypes,to determine whether A1S₁969α recombinant protein can stimulate the body to produce specific antibodies.6.Design four different protein dose to immune mice,then detect bacteria amount in blood in 12 hour after infection,statistics survival rate of mice in 7days after infection,also detect inflammatory factor in serum after infection,screening the optimal immune dose.7.Compare A1S₁969α recombinant proteins with freund’s adjuvant to immune rabbit and collect the immune serum to obtain IgG polyclonal antibody for passive immunization.First,A1S₁969α specific Ig G,Acinetobacter baumannii,and neutrophils incubation in vitro.Then through the detection of bacterial clone inmice,inflammatory serum factors and survival mice in immune group to evaluate the protection of A1S₁969α Ig G polyclonal antibody.Result:1.The result of Bioinformatics software analysis,A1S₁969 identity is as high as 98% to other Acinetobacter baumannii strains,and detecting by PCR suggest that 83% Ab strains contained A1S₁969 gene sequence in our country.Signal peptide prediction results showed that the protein N-contains 43 amino sequence length of signal peptide molecules.Protein 3D structure prediction shows that A1S₁969 is similar to the outer membrane protein BamA.2.Cloned A1S₁969α purpose gene,molecular weight size is 1107 bp（136 1242 bp）,and successfully build PGEX-6 p-2-a1s₁969α/ XL1-Blue recombinant genetic engineering strains;The obtained recombinant carrier gene sequencing data is same to the sequence in Genebank.3.PGEX-6 p-2-A1S₁969α/ XL1 – Blue strain was induced by IPTG in different temperature,SDS-PAGE electrophoresis showed that PGEX-6 p-2-A1S₁969α/ XL1 – Blue highly expression of GST-A1S₁969α fusion protein under 30 ℃.After protein purification,A1S₁969α recombinant protein molecular weight is 42.233 k Da,protein purity higher than 95%,N-terminal amino acid residues is same to protein sequences in Genebank,also the bacterial endotoxin in A1S₁969α recombinant protein is qualified.4.ATCC-17978 strains were grown in LB Liquid culture under 37 ℃and draw the growth curve,and found bacteria growing most rapidly during 5 8 h.ATCC 7978 strains were coated on LB plate and carried out the count of 3.0 x 109 cfu/ml when absorbance was 1.0 at 600 nm.Culture ATCC 17978 for 6 hours,infected mice by intraperitoneal injection and Statistics survival rate of mice and Data Analysis suggest that: the LD50 infection dose was 2.5 x 108 cfu,LD80 infection dose was 4.2 x 108 cfu,death peak was from 12 to 24 h after infection.Infected mice with LD50,found that bacteria loadind in blood,heart,liver,spleen,kidney and lung reaching the peak between 6 to 12 hours after infection,and Ab basic completely cleared by the body in 24 h after infection.5.Immune mice with 30μg A1S₁969α protein antigen,Continuous monitoring antibody levels in the mice,found that Ig G can maintain a higher level after immune 63 and mainly antibody subtype was Ig G1.Active immunity dose was divided into 15μg,30μg,60μ g and 120μg per group.After immune 14 days infection each mouse with ATCC-17978 4.2 x 108 cfu,and Statistical survival of each groups.Compared with the mice of control group,ATCC17979 strains loading in blood microbial were significantly decreased,30μg group（p < 0.05）,60μg group（p < 0.01）and 120μg group（p < 0.01）.Proinflammatory factor IL-1β,TNF-α and IL-6 blood were significantly lower than the control group,60μg group（p < 0.01）and 120μg（p < 0.01）.the survival Of mice also increased in high dose immune group,60μg group survival rate was 70%（p < 0.05）,and 120μg group survival rate was 80%（p < 0.01）,so 120μg as the best immune dose group.6.In Passive immunity,the mice of A1S₁969α IgG polyclonal antibody immune group urvival rate was 90%,and compared to controls group the survival rate of mice after infection was obviously increased（p < 0.01）.At the same time,the amount of ATCC-17978 stains loading in t blood,heart,liver,spleen and kidney were significantly lower in immune mice in than the control mice（all of them,p< 0.01）.Also the inflammatory factors IL-1β,TNF-α and IL – 6 in the blood were significantly reduced in immune mice（all of them,p< 0.01）.Therefore A1S₁969αIg G polyclonal antibody Passive immunity can provide rapid immune protection in mice.7.Antibody function test in vitro showed that A1S₁969α specific IgG Antibody can enhance and promote neutrophil phagocytosis of Acinetobacter baumannii.A1S₁969α immune serum of mice can mediate neutrophil phagocytosis 58.68% of the Ab（p < 0.01）,A1S₁969αIgG polyclonal antibody can mediate neutrophil phagocytosis kill 70.09% of Ab（p < 0.01）.Conclusion:1.Successful cloning,expression and purification A1S₁969α recombinant protein,the purity greater than 95%,molecular weight 42.233 k D,and N-end amino acid sequence are same to the design A1S₁969α protein.2.Establish Acinetobacter baumannii infection mouse model,lay a Experimental foundation to screen advantage antiges for Acinetobacter baumannii.3.The preliminary evaluation A1S₁969α recombinant protein immune protection results show that using A1S₁969α recombinant protein in mice after active immunity and passive immunity,can stimulate the body to produce effective immune response,can promote the mice clean acinetobacter baumannii,inhibit the production of proinflammatory factor,and significantly improve the survival rate of mice.4.the research of the structure and function of A1S₁969α protein have not been reported in the word,Through our study we found A1S₁969α protein could provide immune protection in mice after ATCC 17978 infection,have the possibility to become an candidate antigen for acinetobacter baumannii.At the same time,this topic of acinetobacter baumannii systemic infection of mice death model,can be used to as one of the animal models to screen and evaluation candidate antigens of acinetobacter baumannii.