Preparation And Quantitative Measurement Of Recombinant Cladosporium Herbarum Allergen Cla H8

Cladosporium herbarum is one of the most important allergenic fungal species that has been reported to cause severe asthma,which is an indoor as well as an outdoor allergen source with seasonal peaks in late summer and autumn. It is showed that NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) is a major allergen, which is recognized by IgE antibodies of～57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Sensitization to Cla h8 associated with strong weal and flare reactions by skin prick testing. This study focused on the preparation of high purity of the recombinant protein and its identification and quantitative detection of immunity, providing more accurate diagnosis and more effective and safe means of immunotherapy for the fungal allergy sufferers.The total RNA was acquired from C. herbarum culture, then the cla h8 gene fragments amplified by RT-PCR was cloned into vector pET-19b and then transformed to E. coli BL21 Star (DE3)pLysS. After induction,Cla h8 is highly expressed as inclusion body in E. coli BL21 Star (DE3)pLysS. The soluble protein, following purification and renaturation, was obtained.The purity of the protein from the method can reach as high as 98%. The immunological activity is identified by Dot-blotting and Western blotting, showing that the IgE and IgG of the serum form C. herbarum allergic patients can specially react with the recombinant Clah8 (rCla h8), and the immunological activity of rCla h8 is comparable with the native protein. We produced the mice monoclonal antibodies against rCla h8, the resulting clones 3G10 and 1F3 were screened by Western blot for antibody activity. Optimal antibody pairs were performed using anti-rCla h8 mAb as coating antibody and HRP labeled anti- rCla h8mAb as labeled antibody, which optimal concentrations were defined by titration. Standard curve was performed using purified rCla h8. The sandwish ELISA for detecting rCla h8 was obtained, which characterized for its reproducibility, sensitivity and simple operation.we have successfully obtained the recombinant Cladosporium herbarum allergen Cla h8 and established the double antibody ELISA for quantitative rCla h8, which will lay the foundation for the standardization of fungal allergen vaccination, as well as for diagnosis and treatment.