犬小孢子菌丝氨酸水解酶家族1蛋白的亚细胞定位分析  

Subcellular localization of serine hydrolases 1 protein in Microsporum canis

作　　者：张芙蓉[1] 郭春梅[2] 谭灿 刘洋 徐宇 杨国玲[6] Zhang Furong;Guo Chunmei;Tan Can;Liu Yang;Xu Yu;Yang Guoling(Department of Dermatology,Dalian Friendship Hospital,Dalian 116001,Liaoning,China;College of Basic Medical Sciences,Dalian Medical University,Dalian 116044,Liaoning,China;Department of Dermatology,Chengdu Second People′s Hospital,Chengdu 610017,China;Department of Dermatology,Hangzhou Third Hospital,Hangzhou 310009,China;Department of Dermatology,Piduqu An Jing Coutryside Hospital,Chengdu 611731,China;Department of Dermatology,The First Affiliated Hospital of Dalian Medical University,Dalian 116011,Liaoning,China)

机构地区：[1]大连市友谊医院皮肤科,116001 [2]大连医科大学基础医学院,116044 [3]成都市第二人民医院皮肤科,610017 [4]杭州市第三人民医院皮肤科,310009 [5]成都市郫都区安靖镇卫生院,611731 [6]大连医科大学附属第一医院皮肤科,116011

出　　处：《中华皮肤科杂志》2020年第12期998-1003,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金(81071330)。

摘　　要：目的对丝氨酸水解酶家族1(FSH1)蛋白在犬小孢子菌中进行亚细胞定位分析。方法以前期构建的犬小孢子菌FSH1质粒及载体pCAMBIA-LRP-增强型绿色荧光蛋白(EGFP)为模板,PCR扩增FSH1基因及EGFP基因;同时利用SnaBI/KpnI对pCAMBIA-LRP-EGFP质粒双酶切获得载体DNA,将扩增的EGFP基因克隆至酶切好的载体DNA中获得EGFP表达载体;将扩增的FSH1基因及EGFP基因克隆至酶切好的载体DNA中获得融合载体Ptrcp-FSH1-EGFP-Ttrcp。通过根癌农杆菌介导的遗传转化方法,用重组质粒转化犬小孢子菌,使融合基因FSH1-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,利用激光共聚焦显微镜观察融合蛋白的细胞定位。结果成功构建根癌农杆菌转化系统及犬小孢子菌EGFP表达载体;融合基因FSH1-EGFP在犬小孢子菌中获得整合型表达。激光共聚焦显微镜显示FSH1-EGFP融合蛋白的荧光信号呈颗粒状或团块状集中于犬小孢子菌的细胞质及细胞核中。结论FSH1-EGFP融合蛋白成功定位于犬小孢子菌的细胞质及细胞核中,为进一步明确犬小孢子菌FSH1基因的功能及致病机制奠定了基础。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.

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 

分 类 号：R756.1[医药卫生—皮肤病学与性病学]