Development Of The Colloldal Gold Immunochromatographic Strip For Diagnosis Of Brucella Serum And Lyme Disease

Brucellosis is a chronic bacterial zoonotic disease chronically caused by the genus Brucella which can invade the organism. Brucella can infect humans, various domestic animals and wildlife, which seriously affects the development of animal husbandry and safety of public health. Therefore, Developing a fast and effective methods for diagnosis of this disease becomes urgent. Currently, many methods of the disease have been successfully established by domestic and overseas scholars, some of which turned to be effective. However, these methods are not suitable for animal husbandry in grassroots as they have some disadvantages such as higher price, complex operation, high specialization, and so on. Therefore, it is essential to establish a fast diagnostic method with cheap price, higher sensitivity, convenient, simple operation, and suitable for animal husbandry in grassroots. Our study aims to establish a method for rapid detection for Brucella, and the detection is based on the surface protein of Brucella, it is labelled with Colloidal Gold, which will be a good tool for preliminarily, rapid, effective and simple screening of Brucella.Lyme disease is a bacterial zoonotic disease caused by Borrelia persica via ticks transmitting, which can damage multiple organs. Most of humans and animals infected by Borrelia burgdorferi will experience multiple of periods, and each of the periods show different clinical symptoms, which are easily confused with those of other illnesses. The disease has seriously endangered the safety of public health, so the studies on Lyme disease had caused the general attention. Due to the characteristic that some proteins on the outer membrane of Borrelia burgdorferi have certain immunogenicity, the Borrelia burgdorferi antigen protein for Lyme disease was obtained by the method of prokaryotic expression in the study, and its molecular and serological detection methods are also preliminarily investigated.The main research result concerning Brucellosis and Lyme disease, which are Bacterial zoonotic diseases highly erupt in Xinjiang, are reported as follow:1. The Brucella BP26, VirB5, SP41and OMP31Surface membrane proteins were purified and validated. The four Brucella Surface membrane proteins with high immunogenicity and high specificity were preserved in laboratory, and were purified by affinity chromatography. Through testing the WB experiment and ELISA experiment, we verified their immunogenicity. The result of western blot showed that the four recombinant proteins had high immunogenicity and reactionogenicity, which laid a solid foundation for preparation of immunochromatographic strip for Brucellosis.2. The immunochromatographic strip for Brucellosis was synthesized. The purified BP26, VirB5, SP41and OMP31were used as detection antigen. Reducing hydrogentetrechloroaurate with sodium citrate method and optimizing the reaction conditions, the immunochromatographic strip was ultimately synthesized based on Brucella protein VirB5.166Clinical samples were detected by the synthesized Rose Bengal Plate Test, tube agglutination test and immunochromatographic strip, the results showed that positive rates are34.3%(57/166),36.7%(61/166) and36.1%(60/166) respectively. Coincidence rate between the immunochromatographic strip and Rose Bengal Plate Test was100%, the coincidence rate between the immunochromatographic strip and tube agglutination test was98.4%. The whole testing process can be finished within10min. The immunochromatographic strip has the characteristics of high accuracy, high sensitivity and simple to operate.3. The Outer membrane protein A (OspA) and Outer membrane protein B (BmpA) of Borrelia burgdorferi standard strain for Lyme disease were expressed and purified,and their antibody was synthesized.First, the Lyme disease borrelia burgdorferi OspA and BmpA Prokaryotic Expression System was established, then we purified the OspA and the BmpA by applying the affinity chromatography, after that, the WB method was used to test their reactogenicity, thus the recombinant protein of Borrelia burgdorferi with high expression were successfully obtained, which laid a foundation for further preparation of immunochromatographic strip for Lyme disease.4. The immunochromatographic strip for rapid diagnose for Lyme disease caused by Borrelia burgdorferi was synthesized. The method of preparation of Colloidal gold, the qualitative identification for Colloidal gold particles and the labelling conditions of antibody was in correspondence with the preparation of immunochromatographic strip for Brucellosis. Using the purified OspA and BmpA as detection antigen respectively, the colloidal gold labled immunochromatographic strip was synthesized. And also we made evaluation of their specificity and sensitivity. The results showed that standard positive serum for rabbit Lyme disease detected by immunochromatographic strip was1:200. Among the20copies of suspected Lyme disease sheep serum detected by immunochromatographic strip, two of them were positive. The coincidence rate between the result and the western blotting is100%. The whole detection process can be completed in just10min. The immunochromatographic strip can be kept in refrigerate at4℃for more than half a year.[Conclusion]:Four outer membrane proteins of Brucella were successfully expressed and purified and immunochromatographic strip was synthesized using surface membrane proteins VirB5as diagnostic antigen. The prokaryotic expression system for gene OspA and BmpA were successfully constructed and immunochromatographic strip was synthesized using OspA and BmpA as diagnostic antigen.