Study On The Human Brucellosis Diagnostic Techniques Based On Multiple Epitopes Recombinant Protein

Currently, brucellosis is a reemerged zoonotic infectious disease with a rising incidence in recent years. Approximately 500,000 people annually around the world are affected, and the incidence is increasing year by year in China, over 60,000 human cases were identified in 2015. Brucellosis will not only cause infected animal abortion, tremendous economic losses to the livestock industry, but also will bring great harm to human health. After human infection, the patients may have fever, hepatosplenomegaly, osteoarthritis, hematological changes and other clinical symptoms, serious human brucellosis can lead to pregnant abortion, neonatal death or birth weight reduction. For brucellosis diagnosis, prevention and supervision has attracted extensive attention around the world. Existing brucellosis diagnostic methods have more or less disadvantages, such as complicated to operate, time-consuming, low sensitivity, easy to cross-reactivity and so on. To explore simple, rapid and sensitive diagnostic methods for diagnosis of brucellosis has a very important significance for early diagnosis and early treatment that reducing medical burden and economic loss. In this study, we intend to use immunoinformatics technology to predict B cell epitopes in the major outer membrane proteins of Brucella, then combine with fusion protein technology, the epitopes of Brucella outer membrane proteins are fused to prepare a fusion protein which as a new antigen with capable of recognizing antibody of ant-Brucella specifically. First, the fusion protein as diagnostic antigen to established an indirect ELISA for diagnosis of human brucellosis. Secondly, the fusion protein coupled with the magnetic nanoparticles using immune magnetic bead separation technology to prepare a bioprobe which can recognize Brucella antibodies specifically,combines with quantum dot fluorescence probe which are prepared by quantum dots conjugated SPA, to establish a rapid diagnostic technology of brucellosis. The main contents of this paper consists of four parts:Part ⅠDesign and evaluation of the Brucella fusion protein.1. Based on a literature researvh, four immunodominant outer membrane proteins of Brucella were selected successfully after BLAST analysis: OMP31, OMP16, OMP2 b and BP26.2. Based on immunoinformatics parameters(hydrophilic, flexible, surface accessibility and β turn) prediction and linear B-cell epitope prediction tools, 15 overlapping B-cell epitopes were predicted in the selected four outer membrane proteins.3. The predicted 15 B cell epitopes were concatenated by a linker peptide, and constructed a multiple epitope fusion protein of Brucella successfully. This fusion protein contains a total of 440 amino acids, and its molecular weight is about 60 KDa. The antigenicity of this recombinant fusion protein was predicted, and the result indicated that the fusion protein was a good antigen.Part Expression, purification and evaluation of Brucella fusion proteins. Ⅱ1. To use the genetic engineering principles, based on the amino acid sequence of the self-designed fusion protein, we deduced and optimized codons, to make codons fit for E. coli expression. A total length of 1338 bp fusion protein gene was synthesized successfully, and connected into the expression vector p ET-28 b. After that, the recombinant plasmid was transferred into BL21(DE3) successfully, the fusion protein were expressed through the E. coli expression system, and it is an inclusion body protein, after a denaturation and renaturation, the inclusion body was purified using two commonly protein purification methods which is nickel ion affinity chromatography and anion exchange chromatography the concentration of purified protein is 1.217 mg / m L, the purity is more than 90 % by SDS-PAGE gel electrophoresis analysis.2. The fusion protein was used as an antigen, to prepare a vaccine and immunized mice, the vaccinated mice were induced to product antibodies successfully, analysis of serum antibody subtype, the mainly antibody is Ig G, by flow cytometry analysis of T cell subsets in mouse splenocytes, the percentage of CD4+, CD8+ and the CD4+/CD8+ ratios were increased, indicating that the fusion protein mice induced a strong immune response, the fusion protein has a strong immunoreactivity.3. By indirect ELISA method, the fusion protein can be identified by rabbit antiBrucella 16 M serum and chicken anti-Brucella 16 M serum, proved the fusion protein has good antigenicity, but not reacted with f rabbit anti-E. coli O157: H7 serum and rabbit anti-monocytogenes Listeria serum which proved that the fusion protein are specific.Part ⅢThe establishment and evaluation of indirect ELISA diagnostic methods for human brucellosis.1. Used the Brucella multi-epitope recombinant protein as an diagnostic antigen, to establish an indirect ELISA method for diagnosis of brucellosis and the reaction conditions of the indirect ELISA methods were optimized, including coating concentration and coating time of the antigen, the types of blocking solution and blocking time, dilution and incubation time of the primary body, dilution and incubation time of the secondary antibody, chromogenic substrate time and so on. The specificity, repeatability and sensitivity of optimized IELISA was evaluated, and obtained good results.2. 248 serum samples were detected by the optimized indirect ELISA method, and based on the OD450 values, a ROC curve analysis was performed, the area under the curve is 0.9409, Cut-off value is 0.3865, sensitive and specificity was 88.89 % and 85.54 % respectively, positive predictive value and negative predictive value was 93.75 % and 89.42 % respectively, and as a diagnostic antigen, to compare with the whole-cell antigen, the fusion protein has a better diagnostic sensitivity and specificity.Part ⅣEstablishment and evaluation of a rapid diagnostic methods of brucellosis.1. Used commercially available nano-beads and fluorescent quantum dots to couple with the Brucella multiple epitope fusion protein and SPA respectively, the coupling rate was optimized, we prepared magnetic nanoprobe and quantum dot fluorescent probe.2. Based on the prepared two bioprobes, a new diagnostic method of brucellosis was established, and the conditions of this method were optimized. The optimized method requires only 100 min to complete a diagnosis, and compared to the indirect ELISA method, it can decrease the diagnostic time significantly.3. 248 serum samples were detected by the optimized method, and based on the fluorescence intensity values, a ROC curve analysis was performed, the area under the curve is 0.970, cut-off value is 150.4, sensitive and specificity was 96.15 % and 94.12 % respectively, positive predictive value and negative predictive value was 95.89 % and 94.12 % respectively, and as a diagnostic antigen, to compare with the whole-cell antigen, the fusion protein has a better diagnostic sensitivity and specificity. To compare with the indirect ELISA, the new method has better sensitivity, specificity, positive predictive value and negative predictive value, this method has the better diagnostic effect.In summary, in our study, we used the immunoinformatics technology and fusion protein technology, a Brucella multiple epitope fusion protein was constructed and expressed. based on the fusion protein, a sensitive and specific indirect ELISA diagnostic method of human brucellosis was established, combined with immune magnetic bead separation technology and quantum dots labeling technology established a rapid diagnosis of brucellosis, it has important significance to develop the brucellosis diagnostic kits which have independent intellectual property rights of China.