犬小孢菌PQ-LRP蛋白的定位研究

Localization of PQ-LRP protein in Microsporum canis

目的用增强型绿色荧光蛋白(EGFP)标记犬小孢子菌PQ-LRP蛋白,明确PQ-LRP蛋白在犬小孢子菌中的定位。方法提取犬小孢子菌总RNA,反转录为cDNA作为模板,PCR扩增PQLRP基因,将PQ-LRP基因与EGFP基因构成融合基因,连接到pCAMBIA 1300质粒载体.采用根癌农杆菌转化法对犬小孢子菌进行转化,使融合基因LRP-EGFP在真菌通用启动子(Ptrpc)和终止子(Ttrpc)调控下在犬小孢子菌中整合型表达,激光共聚焦显微镜检测融合蛋白的细胞定位。结果成功构建表达载体pCAMBIA-LRP-EGFP,融合基因LRP-EGFP在犬小孢子菌中获得整合型表达。激光共聚焦显微镜下LRP-EGFP的荧光信号呈颗粒状或团块状集中于犬小孢子菌的细胞膜上。结论LRP-EGFP融合基因在犬小孢子菌中成功表达,且PQ-LRP蛋白定位于犬小孢子菌细胞膜上。

Objective To determine the location of PQ-LRP protein in Microsporum canis using the enhanced green fluorescent protein (EGFP) as a marker. Methods The total RNA was extracted from Microsporum canis, and reversely transcribed into cDNA. The PQ-LRP gene was amplified by PCR using the above cDNA as the template. The fusion gene of PQ-LRP gene and EGFP gene was linked to the plasmid pCAMBIA 1300. Microsporum canis was subjected to Agrobacterium tumefaciens-mediated transformation, in order to achieve the integrated expression of the fusion gene LRP-EGFP in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc. Laser-scanning confocal microscopy was conducted to determine the cellular localization of the fusion protein. Results The expression vector pCAMBIA - LRP - EGFP was successfully constructed, and the fusion gene LRP - EGFP was expressed integratedly in Microsporum canis. Laser-scanning confocal microscopy showed that fluorescence signals of LRP-EGFP were concentrated on the cell membrane of Microsporum canis, giving a granular or cluster-like appearance. Conclusion The infusion gene LRP - EGFP can be successfully expressed in Microsporum canis, and PQ-LRP protein is located on the cell membrane of Microsporum canis.