日本血吸虫mir-125b靶向调控宿主CD276的鉴定研究  

IDENTIFICATION OF PATHOGEN mir-125b TARGETING MURINE CD276 IN MICE INFECTED WITH SCHISTOSOMA JAPONICUM

作　　者：王立辉[1,2] 朱丽慧[2] 刘军涛[2] 夏天奇[2] 陈永军[2] 李建军[1] 程国锋[2] WANG Li-hui ZHU Li-hui LIU Jun-tao XIA Tian-qi CHEN Yong-jun LI Jian-jun CHENG Guo-feng(Tianjin Agricultural University, Tianjin 300384, China Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, CAAS, Shanghai 200241, China)

机构地区：[1]天津农学院,天津300384 [2]中国农业科学院上海兽医研究所农业部动物寄生虫学重点实验室,上海200241

出　　处：《中国动物传染病学报》2017年第4期34-39,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(31502056和31672550);中国农业科学院科技创新工程

摘　　要：通过荧光素酶实验验证宿主CD276为日本血吸虫mir-125b的靶基因,并分析感染血吸虫后宿主CD276基因在不同组织中的表达情况。利用PCR扩增mir-125b与CD276互作的核酸序列,并将目的片段克隆至表达荧光素酶报告基因质粒的下游。将重组质粒转染到HEK293T细胞内,检测荧光素酶报告基因表达。进一步利用荧光定量PCR技术检测小鼠巨噬细胞在转染血吸虫mir-125b mimic后,靶基因CD276的表达情况,同时检测感染血吸虫不同时间段、小鼠不同组织中CD276的表达情况。荧光素酶实验表明转染mir-125b mimic和重组质粒后细胞的荧光素酶报告基因的表达极显著降低(P<0.01),提示mir-125b可与CD276相关区域互作,抑制报告基因的表达;血吸虫mir-125b mimic转染小鼠巨噬细胞后导致其宿主CD276基因的表达降低,进一步提示宿主CD276可能为血吸虫mir-125b靶基因;荧光定量PCR分析表明感染日本血吸虫后的不同感染时期,小鼠淋巴细胞、肝脏及脾脏中的CD276的表达水平显著低于对照组(P<0.01),进一步表明虫源性mir-125b在体内影响宿主CD276基因的表达。日本血吸虫mir-125b可能靶向调控宿主CD276基因的表达,在虫体与宿主互作中发挥重要调控功能。To validate Schistosoma japonicum（S. japonicum） mir-125b targeting murine CD276 and analyze the expression of CD276 in different tissues of mice infected with S. japonicum, we predicted in our previous study that Schistosoma japonicum mir-125b might target murine CD276. Here, we used PCR to amplify the putatively interacted regions of murine CD276 and cloned the amplified fragment into the downstream of a Gaussia luciferase vector（p Gluc-Basic）. Then, the recombinant plasmids mir-125b mimics and anti-sense mir-125b were transfected into HEK293 T cells and the luciferase activities were measured by using a dual luciferase reporter system. In addition, the expression of CD276 in vitro cultured murine macrophages was also determined in quantitative real-time PCR upon mir-125b mimics transfection. Finally, the expression of CD276 in different tissues of mice infected with S. japonicum was also determined in quantitative real-time PCR. The putatively interacted region of CD276 was obtained and the corresponding recombinant plasmid was successfully constructed. A significant down-regulation of luciferase reporter activity was observed upon transfection of mir-125b mimics into HEK293 T cells（P 〈 0.05）. Transfection of mir-125b mimics into murine macrophages led to the down-regulation of CD276. In addition, the expression of CD276 in lymphocytes, livers, and spleens of mice infected with S. japonicum was also down-regulated（P〈 0.05）. Our results indicated that S. japonicum mir-125b directly target murine CD276 during schistosome infection, suggesting that mir-125b might play an important regulatory role in parasite-host interactions.

关 键 词：日本血吸虫 mir-125b CD276 功能 

分 类 号：S852.735[农业科学—基础兽医学]