| Alicyclobacillus are Gram-positive bacteria with thermoacidophilic characteristic which can cause the quality decline of various fruit juice,severely affecting the healthy development of fruit juice industry.To develop a sensitive and accurate way for the convenient and rapid identification of Alicyclobacillus in fruit juice is always the main focus of researchers and main concern of industrialists.The aim of this work is to establish immunoassays for the detection of Alicyclobacillus.The cell wall protein profile of Alicyclobacillus was studied to search for the specific potential targets on its surface.And one of the identified proteins was chosen as antigen for preparing monoclonal antibody.Therefore,the sensitive and specific identification of Alicyclobacillus in apple juice could be achieved.The main research contents and results were as follows:(1)Alicyclobacillus acidoterrestris DSM 3923 was chosen and the bacterial cell wall proteins were first extracted.Some of them with immunogenicity were identified using immunoproteomics technique.The results showed that there are more than 200 proteins in the cell wall of A.acidoterrestris DSM 3923 which mainly distributed in the range of 10-75 k Da.Typically,proteins in the range of 35-75 k Da showed relative lower immunogenicity compared with proteins in the range of 10-35 k Da which exhibited high immunogenicity.Among the 22immunogenic proteins picked,eighteen of them were successfully identified and classified into 12 categories including 2 novel immunogenic proteins.Except for one protein with unknown function,the rest of them could involve in the carbohydrate and energy metabolism,transmembrane transportation of substance,catalyzing the redox reaction,responding to oxidative stress,maintaining cell growth and polypeptide biosynthesis.(2)To obtain 12 purified immunogenic proteins,the primers were designed based on their coding sequences.And the corresponding fusion proteins were obtained by prokaryotic expression technique and their immunogenicity was further evaluated.The results showed that the coding sequence of these proteins can be successfully amplified using the rational designed primers.The length of resultant products was consistent with the prediction and no base mutation detected in the products.Under the induction of isopropyl-beta-D-thiogalactopyranoside(IPTG),the coding sequence can express efficiently in Escherichia coli.Four resultant fusion proteins existed in soluble form and others are inclusion body.These proteins were then purified and their immunogenicity was testified.Except for malate dehydrogenase,other 11 proteins showed strong immunoreaction with the polyclonal antibody of A.acidoterrestris.(3)The hypothetical protein N007?08970 was chosen as antigen for immunizing BALB/c mouse to obtained monoclonal antibody.The titer and specificity of purified antibody were further evaluated.The results showed that all six mice could produce high titer antisera after four times intraperitoneal immunization.One mouse was then picked for cell fusion and seven positive hybridoma cell lines were finally screened.The cell line,1G10,was injected into abdominal cavity to induce the generation of ascites for monoclonal antibody production.High purity antibody was obtained after Protein G affinity chromatography purification.The concentration and titer and isotype of the antibody were determined to be 2.28 mg/m L,1:80000 and Ig G1-kappa,respectively.Strong immunoreaction between the prepared monoclonal antibody and antigen was revealed by western blot analysis.Moreover,the antibody could specifically identify the corresponding antigen target in the cell wall of A.acidoterrestris,indicating the monoclonal antibody possesses high specificity.(4)To assemble an indirect sandwich enzyme linked immunosorbent assay for the detection of Alicyclobacillus in apple juice,the monoclonal antibody and polyclonal antibody of A.acidoterrestris were used as capture antibody and detection antibody,respectively.And the performance of this assay was also evaluated.The results showed that the optimum concentrations of monoclonal antibody and polyclonal antibody used in this method were 2.5?g/m L and 2?g/m L,respectively.The optimum dilution of horseradish peroxidase labeled antibody(HRP-Ab)was 1:4000.Incubating at 4°C overnight was suitable for antibody adsorption onto the microplate's surface.The excess active sites could be blocked by 5%(v/v)skimmed milk at 37°C for 2h.The most suitable reaction times between bacteria antigen and antibody as well as antibody and HRP-Ab were 60 min.The optimum chromogenic reaction time was 15 min.Under these conditions,the performance of this assay on the detection of A.acidoterrestris could reach the best.The limit of detection(LOD)of this method was as low as 5.4×10~2 CFU/m L.And this assay could efficiently differentiate between target and non-target bacteria.This approach was highly stable and repeatable which can detect A.acidoterrestris in apple juice at the contamination level of 1 CFU/m L within 22h.(5)With the help of monoclonal antibody and polyclonal antibody,a colorimetric and fluorescence dual-mode immunoassay for the detection of A.acidoterrestris was developed.This system used p-phenylenediamine as the substrate of HRP and fluorescein for fluorescence signal generation.When the concentration of two antibodies were both 2.5?g/m L and the dilution of HRP-Ab was of 1:5000,the developed approach showed best performance in the detection of A.acidoterrestris.The LOD of two modes were both 4.8×10~2 CFU/m L.This method was highly specific that can accurately identify A.acidoterrestris in apple juice at the contamination level of 1 CFU/m L within 24h. |
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