Study Of Electrochemical Immunosensor For Mycobacterium Bovis

Bovine tuberculosis (BTB) is a chronic human zoonotic diseases mainly caused by Mycobacterium tuberculosis. It has been listed as a Class B animal diseases by The World Organization for Animal Health (OIE) and a second class of animal disease in China. There is no effective early diagnostic reagents and protective vaccine against the disease.Bovine tuberculosis seriously affects the health of animals, and brings huge economic losses to the livestock industry. The disease poses a threat to human health, because it can be transmitted to human through dairy products and other ways. So it is of great significance to find a fast method to detect bovine tuberculosis for the prevention and control of TB.In recent years biosensors have been developed as new technology for rapid detection of pathogenic factors. Such detection method is less time-consuming, highly sensitive and specific. The immunosensor, a particular biosensor with combination of electrochemical magnification and immunologic specificity, is not only electrochemically sensitive but also target-recognition specific. Therefore, this experiment successfully isolated and purified REC fusion protein of Bovine tuberculosis and developed the corresponding McAb. Two electrochemical immunosensors were developed based on RCE and M. bovis McAb for Bovine tuberculosis diagnosis.The main contents and results:1. The expression and purification of fusion protein RV3872-cfp10-esat-6 (hereinafter referred to as RCE) of Mycobacterium bovisThe RCE protein is expressed in Es cherichia coli (Ecoli BL21) by expression vector pET-28a-RCE, and purified by the Ni-NTA affinity chromatography. The defined RCE protein concentration was 1.3910mg/ml by Brandford method. The fusion protein was proven of good antigen activity by Western blot. 2. The preparation of Mycobacterium bovis monoclonal antibodiesBalb/c mice were immunized by RCE antigen, and three cells that can secrete monoclonal antibodies were screened after five cell fusion by hybridoma technology named 5E5,5D5 and 5D7. Monoclonal antibody ascites had been prepared by intraperitoneal injection of 5E5 cells into Balb/c. Monoclonal antibodies with high purity were acquired by Bitterness-sulfuric acid purification of monoclonal antibody ascites ammonium. The McAb concentration measured was 3.089mg/ml by UV assay. By Western blot analysis, the monoclonal antibody exhibited good specificity, which could be applied for development of immunesensors to diagnosing corresponding antibody.3. RCE Mycobacterium bovis Sensor fusion proteinBecause of its high sensitivity, simple operation, fast detection speed electrochemical immunoassay sensing shows a good prospect. By text layer self-assembly technology, the positively charged thionine (Thi) dyes and negatively charged gold nanoparticles (nano-Au) were bond to Nafion-MWCNT film modified glassy carbon electrode (GCE) surface through electrostatic interactions and covalent bonding to prepare a nano-Au/Thi/Nafion-MWCNT sensitive film. Then the RCE protein fixed on the electrode surface by means of its adsorption to nano-Au thus established electrochemical immunosensor based on Mycobacterium bovis protein RCE. The sensor showed great sensitivity, specificity, reproducibility, etc. For immuno-electrode characterization and qualitative judgement, the CV method were used to measure the decreasing proportion of reduction peak current before and after immune response to assay antibodies in serum samples.The optimum RCE fixation concentration was determined as 500μg/ml after trials and the optimum time of reaction was 60min. The immunosensor showed excellent response to polyclonal antibodies and the reduction peak current rate k diminished with the decrease of antibody concentration. The linear regression equation was k= 3.8751nC+71.625, R= 0.9976 while the Abs were diluted to 1:80-1:2560. The immunosensor exibits good sensitivity (when RCE antibody was diluted 20480-fold, the Elisa result is negative, but the immunosensor result is positive), specificity (k value (47.1%) induced by RCE antibodies than the other k values caused by several bacteria), repeatability (within-batch RSD=1.51%,between-batch RSD=2.97%). The criteria to determine positive/negative charicteristics of this immunosensor was preliminary established,with the k>15.4% as positive and k<15.4% as negative. The assayed results was 100% consistant with the ELISA tests.This research indicated the applicability of this immunosensor for detection of Bovine tuberculosis, providing the foundation for further clinic implement.