Identification Of The Target Protein Of Monoclonal Antibody G9in Nosema Bombycis

The microsporidia are an unusual group of eukaryotic, obligate intracellular parasites, which have attracted the curiosity of biologists for more than100years. Like many other intracellular parasites, microsporidia are highly specialized and have an extremely sophisticated and unique infection mechanism along with other adaptations to life inside the host cells. Nosema bombycis is a parasite of Bombyx mori, the pathogen of silkworm pebrine disease, which can lead serious losses to silk industry. Therefore, how to detect and prevent N. bombycis at early stage becomes an important and difficult task for sericulture.In1870, Louis Pasteur set up the female moth microscopy assay to detect N. bombycis, this method has been used for more than150years, and contributes a lot in preventing the pebrine disease in silkworm eggs production. The history of N. bombycis detection experienced the following phases:microscopic examination, molecular biology detection, immunology detection. Following the traditional female moth microscopy methods, the use of staining method improved the detection rate of N. bombycis. Transmission electron microscope can observe the ultrastructure of Nosema bombycis, which can identify the species of microsporidia, according to the thickness of the spore wall, polar tube coils and angle. With the development of molecular biology techniques, PCR and LAMP technology were constructed to detect Nosema bombycis.In recent years, according to the different antigens of the spore wall, the immunological detection method was established. Immunochromatography combines the advantages of chromatography and immunoassays, by which the reaction between antibody and antigen occurs after chromatographic separation through a nitrocellulose membrane using capillary flow. Rapid test strip played a significant role in the field of medicine, pathogen diagnosis and food safety. In2009, Our laboratory developed the fast and simple detection strip for Nosema bombycis spores by use of monoclonal antibodies G9(MAb G9) and rabbit polyclonal antibody both against Nosema bombycis. The strip was used to detect spores inside the infected larva, or female moths. However, the specificity and the sensitivity of the test strip can’t meet the requirements in production. Therefore, how to improve the specificity and sensitivity of the test strip to be suitable for production applications is very important.Based on the above, this paper analyzes the specificity and sensitivity of the test strip, and we try to confirm the target protein of MAb G9and then to refine the strip to improve the specificity and sensitivity of the test strip. The results are as follows:1. Analysis of sensitivity and specificity of the detection strip for Nosema bombycisWe found that the target of test strip detection is present in the supernatant after the alkali treatment. Sensitivity and specificity experiments were performed to analyze the detection performance of the test strip. In specificity experiment, as Bombyx mori nuclear polyhydrosis virus, Nosema antheraeae, Vairimorpha bombycis were detected, nonspecific signal was observed only in the sample of Bombyx mori nuclear polyhydrosis virus. In sensitivity experiment, we first treated the spores with0.1mol/L KOH solution, and then the alkali mixtures were detected by test strip. Respectively, the total number of spores is10,107,106,105. The detection signal appeared when samples of108,107spores were detected, the experiment was repeated twice, and yielded same results. Using the Bradford method to measure the protein concentration of the supernatant of the samples of108,107spores treated by alkali solutionthe results are1.76μg/μL and0.17μg/μL respectively, which indicates the lowest protein concentration that can be detected with the test strip is0.17μg/μL.2. Optimization of alkaline treatmentThis chapter we analyze the supernatant proteins(alkali-soluble proteins) after spores were treated by alkali solution.. First of all, the alkali-soluble proteins ware analyzed by SDS-PAGE. Using LTQ to analyze the main bands of alkali-soluble proteins, including the30kDa band, the results show that SWP1is the main component of alkali-soluble proteins. Analyzing alkali-soluble proteins under different alkaline solution, concentration of alkaline solution, time, temperature and pH, we found that different alkaline solution has no impact on composition of alkali-soluble proteins. As the concentration increases, the protein bands also become rich. Gradient processing time analysis of alkali-soluble proteins found that the quantity of protein bands does not increase after105min treatment. As to the temperature, a new protein band around15kDa appeared under60℃treatment compared to other temperatures" treatment. After alkali-soluble proteins were extracted with0.1mol/L KOH solution, then the pH of the solution contained the alkali-soluble proteins were adjusted to8,9,10, the following SDS-PAGE indicated that changes in pH did not affect the stability of the protein.. Alkali-soluble proteins extracted from spores of different sources have different patterns in SDS PAGE. Through research in this chapter, we know the composition of the alkali-soluble proteins and the effect of different conditions to alkali-soluble proteins at a certain extent. This study provided the basic experimental data for searching for the target protein of MAb G9.3. Identification of the target protein of monoclonal antibody G9in Nosema bombycisIn this chapter, we designed and carried out immunoprecipitation and Western blotting experiments to find the target protein of MAb G9. Immunoprecipitation experiments between alkali-soluble proteins and MAb G9shows that MAb G9precipitated a30kDa protein from the alkali-soluble proteins, identified as SWP1by LTQ MS analysis. To further validate the results of the LTQ, after MAb G9and alkali-soluble proteins immunoprecipitation, Western blotting was carried out with anti-rSWP1. The result shows that anti-SWP1detected30kDa bands, and&the size is same with SWP1, the main band of alkali-soluble proteins. At the same time. Western blotting between anti-rSWP1and MAb G9was carried out. ELISA experiment of anti-rSWP1and alkali-soluble proteins, MAb G9and alkali-soluble proteins were also performed.The results of two experiments above are same with the results obtained in immunoprecipitation. Finally, we come to a conclusion that is MAb G9against SWP1. In the future, we will improve the specificity and sensitivity of the test strip by use high titer antibody against SWP1as capture line in test strips...