Cloning Of Pigment Fluorescent Protein Gene And Expression In Pichia Pastoris

Green fluorescent protein has excellent fluorescence activity. And it takes as a reporter gene for analyzing protein localization, mobile and interactions in a wide variety of experimental systems. Pigment protein has a wide of spectral range. GFP fusions with pigment protein can broaden the scope of spectrum testing and also can be used for two color or multiple color markers when excited by light. The fluorescent protein tags enlarge the application in cell biology. Florescence Probe is deserved more and more attention for treatment and location. In addition, Potential biological activities of pigment protein in Cyanobacteria such as antioxidant activity in vitro and as photosensitiser for photo dynamictherapy(PDT) had gained widely concerning.This dissertation includes the following sections:(1) The clone of gfp fusions with actin gene expessed in prokaryote system. The expression vector pCDFDuet-mreb:gfp was constructed and transformed into strain BL21. The fluorescence microscope pictures, SDS-PAGE, fluorescence and absorption spectrum suggest that the fused gene was expressed successfully in strain BL21.(2) The expression of gfp gene and gfp fusions with pcyA gene of phycocyanobilin in pichia pastoris strain GS115.The two recombinant yeast expression vector were constructed based on the two gene sequences cloned, respectively. Then the two expression vectors were transformed into the genome of the pichia pastoris strain GS115by electrotransformation method. The fluorescence microscope pictures, SDS-PAGE, fluorescence and absorption spectrum suggest that the two genes were expressed well in GS115. This reserch preliminarily establishes pichia pastorios expression system and constructs a platform which can express heterologous protein in yeast.(3) The construction of double color fluorescent protein system in Pichia pastorios. First, the yeast expression vector pPIC3.5K:f0:gfP(△SacI):pcyA were constructed.and transformed into the genome of pichia pastoris strain GS115by electrotransformation method. The stain GS115/pPIC3.5K:f0:gfp(△SacI):pcyA was screened by fluorescence microscope pictures and measured by fluorescence and absorption spectrum. Then, another vector pPICZB:abp1:gaf3:ho1which contains phytochrome gene transformed into the genome of engineered pichia pastoris stain GS115/pPIC3.5K:f0:f0fp(△SacI):pcyA, PCR testing and transforments inoculate in medium for protein expression.then fluorescence microscope pictures and fluorescence and absorption spectrum suggesting that the result is failed.