Molecular Cloning, Purification and Immunological Characterization of Major Allergen from German Cockroach (Blattella Germanica) as Arginine Kinase

• Main Authors: Tang-Kuei Lin, 林唐逵
• Other Authors: Lu-Ping Chow
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/96349050788380742919

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 94 === Cockroaches have been reported to be an important cause of asthma for over 40 years. In 1964, Bernton and Brown were the first to report positive skin test responses to cockroach allergen (44%) in a landmark study of 755 allergic patients living in New York. In that study, 13% of patients who otherwise would have been considered nonatopic were sensitized to cockroach allergen alone. There are also 39.4% and 36.4% allergy clinic patients sensitized to Periplaneta americana and Blattella germanica in the study of Taiwan in 1998. So cockroach allergy is also one of the important causes of asthma in Taiwan. The principal domestic cockroach species are Blattella germanica and Periplaneta americana and B. germanica is the most abundant one of indoor species. Previous identified allergens include Bla g 2 (Inactive aspartic protease), Bla g 4 (Calycin), Bla g 5 (Glutathione-S-transferase), Bla g 6 (Troponin), the group 1 cross-reactive allergens Bla g 1 and Per a 1, Bla g 7 and Per a 7 (Tropomyosin), and Per a 3 (Arylphorin). After observation and LC-MS/MS identification of separated proteins of cockroach crude extract on the 2D-immunoblot, we assumed that “Arginine kinase”, an IgE-binding protein, could be a putative novel allergen of German cockroach. In this study, this novel allergen was cloned and the deduced amino acid sequence was a 356 a.a. protein with an acetylated N terminus at Val2. Arginine kinase (AK) of German cockroach showed an extensive similarity with other allergic AK which are from shrimp and Indianmeal moth. Native AK from German cockroach was purified by ammonium sulfate fractionation and anion exchange chromatography. ELISA results showed a 50.79% prevalence of IgE-reactivity among German cockroach allergic patients and AK was able to inhibit 50% of the IgE-binding to total allergens from B. germanica bodies, which showed that AK is an important major allergen. In addition, 17 of 24 AK positive sera completely lost their IgE-reactivity under the SDS-denaturing condition, which indicate the major existence of conformational epitopes (70.8%). After purified P. americana AK was prepared, Dot-blot assay showed the antigenic cross-reactivity between B. germanica and P. americana. In ELISA inhibition assay, we observed that P. americana AK inhibited the IgE-binding to B. germanica AK, which indicated the same IgE epitope between AK from different cockroach species. Then, we demonstrated that AK is not only a major allergen but also a pan-allergen in cockroach species. In the future, we expect that the production of recombinant AK may be helpful to the development of improved forms of clinical diagnosis and immunotherapy for current cockroach allergy diseases.