关键词: 小孢子菌属, 丝氨酸蛋白酶类, 绿色荧光蛋白质类, 犬小孢子菌, FSH1, 亚细胞定位

Abstract: 【Abstract】 Objective To analyze the subcellular localization of family of serine hydrolases 1 （FSH1） protein in Microsporum canis. Methods The FSH1 and enhanced green fluorescent protein （EGFP） genes were amplified by PCR using the previously constructed plasmid containing the FSH1 gene and the recombinant plasmid pCAMBIA-LRP-EGFP as the template; the vector DNA was obtained by double-enzyme digestion of the recombinant plasmid pCAMBIA-LRP-EGFP with SnaBI/KpnI. Then, the EGFP expression plasmid and Ptrcp-FSH1-EGHP-Ttrcp fusion plasmid were constructed by inserting the amplified EGFP gene and EGFP-FSH1 gene into the vector DNA respectively, and identified by PCR and sequencing. The two recombinant plasmids were transformed into Microsporum canis by an Agrobacterium tumefaciens-mediated method, and the gene EGFP and fusion gene FSH1-EGFP were expressed integratedly in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc. The cellular localization of the fusion protein was observed by laser scanning confocal microscopy. Results The Agrobacterium tumefaciens-mediated transformation system and EGFP expression vector in Microsporum canis were successfully constructed; the fusion gene FSH1-EGFP was expressed integratedly in Microsporum canis. Laser confocal microscopy showed that fluorescence signals of the FSH1-EGFP fusion protein were concentrated in the cytoplasm and nuclei of Microsporum canis, with a granular or cluster-like appearance. Conclusion The FSH1-EGFP fusion protein was successfully localized in the cytoplasm and nuclei of Microsporum canis, providing a basis for further clarifying the function and pathogenic mechanisms of the FSH1 gene in Microsporum canis.

Key words: Microsporum, Serine proteases, Green fluorescent proteins, Microsporum canis, FSH1, Subcelluar localization