Secretory Expression Of SPA-EGFP Fusion Protein In Pichia Pastoris And Its Application In Immunodiagnosis

Immunofluorescence assay (IFA) is widely used both in research and diagnosis. Using IFA, it is possible to localize antigens in tissues and individual cells. Antibodies, to which a fluorochrome is chemically added, are extensively used for this purpose. Fluorescein isothiocyanate (FITC) has remained the most popular fluorochrome for IFA until the present time. However, one of the main disadvantages of FITC has been its rapid fading under intense illumination. Furthermore, traditional antibody preparation and conjugation procedure may result in reduced valence of antibody and nonspecific staining. In this study, the fusion protein between enhanced green fluorescent protein and staphylococcal protein A is expressed in Pichia pastoris, which has stable fluorescence activity and the ability to bind IgG, circumventing the disadvantages of traditional fluorescent antibody. The usefulness of this fusion protein is demonstrated through applications for the diagnosis of Autoimmune Diseases and the test of pathogen.Spa-egfp fusion gene was amplified by PCR from recombinant plasmid pEZZ18/egfp and cloned into the Pichia pastoris secretory expression vector pPIC9K. The authenticity of the constructed expression vector pPIC9K/spa-egfp was confirmed by DNA sequencing. After linearized by digestion with Sal I, the recombinant vector was transformed into Pichia pastoris strain GS115 by electroporation. Plating and screening of transformants on MD plates followed by in vivo screening of multiple inserts on YPD-G418 plates were performed according to the Invitrogen manual. Positive transformants were confirmed by PCR and then induced by methanol.