常振策,李忠旬,修江帆,卢成,贾利娜,国果,吴建伟
CHANG Zhence,LI Zhongxun,XIU Jiangfan,LU Cheng,JIA Lina,GUO Guo,WU Jianwei

• 1:贵州医科大学贵州省普通高等学校现代病原生物学特色重点实验室
• 2:贵州医科大学寄生虫学教研室

摘要(Abstract)：

目的:克隆家蝇的Clip-丝氨酸蛋白酶基因(Clip-Msp),并对Clip-Msp在白假丝酵母菌感染后的时空表达模式进行研究。方法:通过基因工程的方法,构建pMD19-T/Clip-Msp克隆载体;采用生物信息学方法,分析Clip-Msp基因的开放阅读框和氨基酸序列及Clip-Msp蛋白的功能结构域;以GAPDH作为内参照,于家蝇三龄幼虫感染白假丝酵母菌后3、12、24及48 h时,采用实时荧光定量PCR方法检测Clip-Msp在不同组织(体壁、脂肪体、血淋巴、唾液腺、肠道、马氏管和气管)中的表达情况。结果:成功构建pMD19-T/Clip-Msp克隆载体,生物信息学分析显示,Clip-Msp基因开放阅读框全长1 524 bp,共编码507个氨基酸、无信号肽、存在跨膜区,属于非经典分泌型蛋白;Clip-Msp蛋白的N端存在一个Clip结构域,C端有一个Tryp_SPc催化结构域,属于单催化结构域Clip-丝氨酸蛋白酶超家族;白假丝酵母菌感染家蝇三龄幼虫后,Clip-Msp基因在不同时间点、不同组织中表达存在差异(P<005),3 h时以血淋巴中表达量增加最高,12 h时以脂肪体、气管、唾液腺及肠道中表达量增加为主、脂肪体内的相对表达量最高,24 h时肠道组织表达增加最明显、脂肪体次之,48 h时血淋巴中表达量高于马氏管及肠道。结论:成功克隆Clip-Msp基因,Clip-Msp基因主要分布在血淋巴、脂肪体和肠道组织。
Objective: To clone Clip-serine protease gene(Clip-Msp) from housefly third instar larva(Musca domestica) and study its temporal-spatial expression pattern afterCandida albicansinfection.Methods: The pMD19-T/Clip-Mspcloning vector was constructed by genetic engineering. The open reading frame and amino acid sequence ofClip-Mspand the function domain of Clip-Msp protein were analyzed by bioinformatics. UsingGAPDHas internal reference gene, qPCR was used to detectClipMspmRNA level in the third instar larvae in different tissues( epidermis, fat body, hemolymph,salivary gland, intestine, malpighian tube and trachea) and at different time points(3 h, 12 h, 24 h and 48 h) after systemic infections withCandida albicans.Results: The pMD19-T/Clip-Mspcloning vector was successfully constructed. Bioinformatics analysis showed that the open reading frame ofClipMspis 1524 bp in length, encoding 507 amino acids.Clip-Msphas no signal peptide, belonging to a non-classic secretory protein. Additionally, Clip-Msp protein has a transmembrane region, a Clip domain at the N-terminal and a Tryp_SPc catalytic domain at the C-terminal, belonging to the Clipserine proteases superfamily with a single catalytic domain. The expression level ofClip-Mspwere different(P< 0. 05) in different tissues at different time points after the infection, peaking in hemolymph at 3 hrs post infection, and upregulated in fat body, trachea, salivary gland and intestine at 12 hrs post infection. The biggest increase in expression level was in fat body. At 24 hrs after infection, there was the highest expression in intestine, followed by fat body; while the expression in hemolymph was higher than that in malpighian tube and intestine at 48 hrs.Conclusion: TheClip-Msp was successfully cloned. TheClip-Mspwas mainly expressed in hemolymph, fat body and intestine.

关键词(KeyWords)： 家蝇;Clip-Msp基因;白假丝酵母菌;基因表达;感染;生物信息学
Musca domestica;Clip-Mspgene;Candida albicans;temporal-spatial expression;infection;bioinformatics

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目（81360254）;; 国家科技支撑计划（2011BAC06B12）;; 贵州省农业攻关[黔科合NY(2014)3054];; 贵州省科学技术基金[黔科合J字(2012)2038]

作者(Author): 常振策,李忠旬,修江帆,卢成,贾利娜,国果,吴建伟
CHANG Zhence,LI Zhongxun,XIU Jiangfan,LU Cheng,JIA Lina,GUO Guo,WU Jianwei

DOI: 10.19367/j.cnki.1000-2707.2020.02.003

参考文献(References)：

• [1]路永鹏，顾冀海，高一夫，等．肽聚糖识别蛋白PGRP-SC在家蝇肠道免疫中的功能分析[J]．昆虫学报，2016,59(3):269-277.
• [2]杨雪，唐婷，王一丽，等．家蝇新型抗菌肽Muscin的基因克隆、表达模式及抑菌活性[J]．昆虫学报，2015,58(6):617-624.
• [3]罗嫚，王宇，胡亚，等．家蝇肽聚糖识别蛋白（PGRP-SA）的克隆表达及细菌结合研究[J]．生物技术通报，2016, 32(8):145-151.
• [4]胡亚，卢诚，魏川川，等．家蝇核糖体蛋白S18基因的克隆及表达模式研究[J]．生物技术通报，2016, 32(6):135-142.
• [5]胡亚，罗嫚，王宇，等．家蝇GNBP3基因克隆及感染白色念珠菌后的表达[J]．环境昆虫学报，2017, 39(3):596-604.
• [6]陈明明，王涛，林小金，等．家蝇抗真菌肽衍生物MAF-1C抗白色念珠菌活性研究[J]．生物技术，2016,152(1):64-69.
• [7] LEE K S, KIM B Y, CHOO Y M, et al. Dual role of the serine protease homolog BmSPH-1 in the development and immunity of the silkworm,Bombyx mori[J]. Developmental&Comparative Immunology, 2018,85(8):170-176.
• [8] XIU J F, WANG T, WANG Y, et al. Histological observation and expression patterns of antimicrobial peptides during Fungal Infection inMusca domestica(Diptera:Muscidae)Larvae[J]. Brazilian Archives of Biology and Technology, 2016, 147(16):1678-90.
• [9]李国辉，周倩，胡朝阳，等．果蝇先天免疫分子机制研究进展[J]．生命科学研究，2015, 19(6):559-564.
• [10]李波，韩文瑜，宋智娟，等．抗菌肽的免疫调节活性及其在动物生产中的应用[J]．中国饲料，2014,4(4):14-17.
• [11]侯成香，覃光星，刘挺，等．昆虫对病原真菌的防御机制研究进展[J]．安徽农业科学，2012, 40(23):11649-11652.
• [12]任菲菲，曹方，杨婉莹．昆虫先天免疫中的肽聚糖识别蛋白（PGRP)[J]．广东蚕业，2014, 48(2):34-38.
• [13] DANA N, HANNAH C, CHESTER J.Staphylococcus aureusin the house fly:temporospatial fate of bacteria and expression of the antimicrobial peptide defensin[J].J Med Entomol, 2013, 50(1):171-178.
• [14]JOYNER C, MILLS M K, NAYDUCH D.Pseudomonas aeruginsainMusca domesticaL:Temprospatial examination of bacteria population dynanmics and house fly antimicrobial responses[J]. PLoS ONE,2013,8(11):e79224.
• [15]吴婧，邢晶晶，王梦婷，等．植物体内的非经典蛋白分泌途径[J]．电子显微学报，2018, 37(2):160-170.
• [16]孙晶，刘欣，张楠楠，等．真核细胞非经典蛋白分泌途径[J]．遗传，2009, 31(1):29-35.
• [17]NICKEL W. Pathways of unconventional protein secretion[J]. Current Opinion in Biotechnology, 2010, 21(5):621-626.
• [18]JIANG H, ROSS J, KANOST M R, et al. Serine proteases and their homologs in theDrosophila melanogastergenome:an initial analysis of sequence conservation and phylogenetic relationships[J]. Gene, 2003, 304(3):117-131.
• [19] CAO X, JIANG H. Building a platform for predicting functions of serine protease-related proteins inDrosophila melanogasterand other insects[J]. Insect Biochemistry and Molecular Biology, 2018, S0965-1748(18):30304-30307.
• [20]YASSINE H, KAMAREDDINE L, CHAMAT S, et al. A Serine protease homolog negatively regulates TEP1 consumption in systemic infections of the malaria vector,anopheles gambiae[J]. Journal of Innate Immunity, 2014,6(6):806-818.
• [21]KANOST MR, JIANG H. Clip-domain serine proteases as immune factors in insect hemolymph[J]. Current Opinion in Insect Science, 2015,10(11):47-55.
• [22]PIAO S, SONG Y L, KIM J H, et al. Crystal structure of a clip-domain serine protease and functional roles of the clip domains[J]. EMBO(European Molecular Biology Organization)Journal, 2005, 24(24):4404-4414.
• [23]TU T T, JI H Q, Guo G, et al. Characterization of the antifungal peptide MAF-1 from the hemolymph ofMusca domesticalarvae and its antifungal activity[J]. Acta Entomologica Sinica, 2012,55(9):1046-1053.
• [24]GRéGOIRE C. Blood coagulation in arthropods III. reactions of insect hemolymph to coagulation inhibitors of vertebrate blood[J]. Biological Bulletin, 1953, 104(3):372-393.
• [25]BUCHON N, POIDEVIN M, KWON H M, et al. A single modular serine protease integrates signals from pattern-recognition receptors upstream of theDrosophilaToll pathway[J]. Proceedings of the National Academy of Sciences, 2009, 106(30):12442-12447.
• [26]JIN X B, WANG Y, ZHU J Y, et al.Expression analysis of antibacterial peptide genes at different development stages ofMusca domestica[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2008, 26(3):187-190.
• [27]李玉欣，亓希武，韩琦，等．家蚕丝氨酸蛋白酶BmHP14基因的克隆与表达模式分析[J]．蚕业科学，2012, 38(5):814-821.
• [28]魏川川，修江帆，胡亚，等．家蝇3种丝氨酸蛋白酶抑制剂（Serpin）基因的克隆、序列分析及表达模式[J]．环境昆虫学报，2017, 39(6):1334-1341.

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