关键词: 德国小蠊, Bla g 2, 变应原, 毕赤酵母, 表达, 免疫学鉴定

Abstract: Objective To express the major allergen of Blattella germanica （Bla g 2） in Pichia pastoris and obtain the soluble protein. Methods The known Bla g 2 gene was used to design the primers which had the restriction enzyme sites. PCR method was applied to obtain the Bla g 2 gene. The gene fragment was then cut and ligated with the Pichia expression vector pGAPZaA, resulting in a recombinant plasmid pGAPZaA-Bla g 2. The linearized pGAPZaA-Bla g 2 was transformed into Pichia pastoris GS115 through electroporation, then screened to positive transformants, and the protein was expressed in YPD medium. Purification of the recombinant protein was achieved by metal （Ni²⁺） chelating affinity chromatography and Western-blotting assay indicated its IgE binding capacity. Results With the expressed recombinant protein, SDS-PAGE showed the presence of the product in the supernatant of the culture with Mr 45 000. After 3 days culture, the recombinant protein occupied 50% of the total proteins in the supernatant. The recombinant protein was purified and Western-blot demonstrated an adequate IgE binding capacity of the product. Conclusion A recombinant protein of Bla g 2 has been obtained, which is soluble in the supernatant and therefore can avoid a process of denaturalization and renaturation of the recombinant.

Key words: Blattella germanica, Bla g 2, Allergen, Pichia pastoris, Expression, Immunoblotting