摘要
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1524 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<0.05),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 after Candida albicans infection.Methods:The pMD19-T/Clip-Msp cloning vector was constructed by genetic engineering.The open reading frame and amino acid sequence of Clip-Msp and the function domain of Clip-Msp protein were analyzed by bioinformatics.Using GAPDH as internal reference gene,qPCR was used to detect Clip-Msp mRNA 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 with Candida albicans.Results:The pMD19-T/Clip-Msp cloning vector was successfully constructed.Bioinformatics analysis showed that the open reading frame of Clip-Msp is 1524 bp in length,encoding 507 amino acids.Clip-Msp has 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 Clip-serine proteases superfamily with a single catalytic domain.The expression level of Clip-Msp were 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:The Clip-Msp was successfully cloned.The Clip-Msp was mainly expressed in hemolymph,fat body and intestine.
作者
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(Key Laboratory of Modern Pathogenic Biology of Guizhou Higher Education Institutions,Guizhou Medical University,Guiyang 550025,Guizhou,China;Department of Parasitology,Guizhou Medical University,Guiyang 550025,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2020年第2期138-144,共7页
Journal of Guizhou Medical University
基金
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]。
