The Expression Of Brucella Wzt And Its Related Properties

Objective:Brucellosis is a infectious disease all over the world.Because its pathogenesis and immunologic mechanism are not clear,the prevention and control of brucella is difficult.Lipopolysaccharide(LPS)is a main virulence factor of brucella,as well as an important immune molecule.Different structures of OPS of different strains lead to different immunologic reaction,but the the constitution of genes for OPS synthesis are conservative relatively.The key genes of LPS synthesis include wbk A?gmd?wzm?wzt?wbk B ?wbk C and so on,and wzt gene is one of the major component gene code of ABC transporter.ABC transporter is important to LPS synthesis.The function of protein Wzt is to hydrolyze ATP and release energy,to transport OPS side chain to outer membrane,and form smooth LPS.The deficiency of wzt can lead to a block of synthesis of LPS,but the properties and its concrete function has not been reported.Explore how Wzt exercise its enzyme activity and study on the feasibility of protein Wzt as an immune candidate antigen as well as the the role in molecular diagnostics can provide experimental basis for further studying its function of LPS synthesis and the feasibility of Wzt as a target to control LPS.Method:This study optimizes the condons of brucella wzt,establishes the prokaryotic expression vector and purifies protein Wzt with the method of affinity chromatography,and then analyses the N-teminal sequence in the way of Edman degradation.According to that ATP can catalyze fluorescence reaction,we detect ATP enzyme activity of Wzt.Forecast the protein model and the key amino acid of enzyme activity with Jpred3 and Phyre2.Mutate the predicted locuses with Site-directed Gene Mutagenesis Kit of Beyotime Biotechnology and detect the ATP enzymatic activity,and make a comparison to protein Wzt.Mix the purified Wzt with freund's adjuvant and immune rabbit and mice BALB/c.After 4 weeks,collect the serum of rabbit and purify Wzt antibody.Detect the specificity of Wzt antibody and use it to detect wzt.Detect proliferation of mice splenic lymphocytes after 4 weeks and detect the titer of Ig G of mice from the first week to the eighth week.Result:Establish the prokaryotic expression vector p ET21b-wzt,and express protein Wzt,it's about 28 k Da and it is the interesting protein.The purification rate of purified protein is 93%,and the recovery rate of Wzt is 2%.This protein has ATP enzyme activity,and 0.05 mg Wzt can catalyze to produce 32872.12 nmol ATP.LYS72 and ASN68 are the key amino acid of its enzyme activity,after these two sites broken,the enzyme reduced to 62% and 67% respectively.The Wzt multiclonal antibody's specifity is well,and we can use it to detect wzt.