树鼩连接黏附分子A的基因克隆及初步功能研究  

Study on Gene Cloning and Preliminary Function of Junction Adhesion Molecule A in Tree Shrew

作　　者：李晓飞[1] 孙晓梅[1] 王文广[1] 匡德宣[1] 陆彩霞[1] 仝品芬[1] 罕园园[1] 李娜[1] 代解杰[1] LI Xiaofei;SUN Xiaomei;WANG Wenguang;KUANG Dexuan;LU Caixia;TONG Pinfen;HAN Yuanyuan;LI Na;DAI Jiejie(Center of Tree Shrews Germplasm Resources,Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming 650118,China)

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所树鼩种质资源中心,昆明650118

出　　处：《实验动物与比较医学》2020年第3期196-203,共8页Laboratory Animal and Comparative Medicine

基　　金：国家自然联合基金重点项目(U1702282);云南省科技人才和平台计划项目(2017HC019);重点实验室运行补助专项(2017DG008);云南省重大科技专项(2017ZF007)。

摘　　要：目的获取树鼩连接黏附分子A(junctional adhesion molecule A,JAM-A)的全长编码序列并进行分子特征分析,探讨JAM-A作为呼肠孤病毒受体入侵树鼩原代肺细胞的作用机制。方法提取健康树鼩组织总RNA,采用反转录PCR和cDNA末端快速克隆技术获取JAM-A基因;Unipro UGENE分析编码氨基酸,使用MEGA 6.0软件最大似然法构建系统发育树;实时荧光定量PCR检测JAM-A在树鼩25个组织和血液中的表达分布情况;于树鼩原代肺上皮细胞上进行受体的特异性抗体阻断处理,然后采用免疫荧光法观察病毒抗原对呼肠孤病毒感染的影响。结果获取了JAM-A基因cDNA全长序列,2962 bp;系统发育树进化分析显示JAM-A基因与人的亲缘关系较啮齿类更近;JAM-A分子广泛表达于树鼩的外周组织,而在呼吸道和消化道表达水平更高。特异性抗体作用于细胞显著降低病毒抗原的免疫荧光积分吸光度值(P<0.0001)。结论首次克隆并分析了JAM-A的基因序列,验证JAM-A分子是呼肠孤病毒入侵树鼩的主要受体,提示树鼩可作为一种新的呼肠孤病毒动物模型。Objective To explore the mechanism of junctional adhesion molecule A(JAM-A)as a reovirus receptor to invade the primary lung cells of tree shrew.Methods Total RNA was extracted from normal tree shrew tissues.JAM-A gene sequences were obtained by reverse transcription-PCR and rapid-amplification of cDNA ends.Translation of amino acids encoded by JAM-A gene were analyzed by using Unipro UGENE software.The phylogenetic tree was constructed by MEGA 6.0 software using the maximum likelihood method.Using real-time fluorescent quantitativePCR to detect the relative expression level of JAM-A in 25 tissues and blood of tree shrew.The specific antibody blocking treatment of the receptor was performed on the primary tree shrew alveolar epithelial cells,and the fluorescence intensity of the virus antigen after reovirus infection was determined.Results The full-length cDNA sequences of JAM-A gene with 2962 bp were obtained.Amino acid alignment and phylogenetic tree analysis showed that the genetic relationship of JAM-A gene in tree shrew was more closer to human than that of rodent.JAM-A molecule was widely expressed in the peripheral tissues of tree shrews,with the more higher expression levels in the respiratory and digestive tract.The integrated absorbance of the immunofluorescence of the virus antigen after the specific antibody was applied to the cells was significantly decreased(P<0.0001).Conclusion The whole sequence of JAM-A gene was retrieved and analyzed for the first time,and the JAM-A molecule was verified to be the main receptor for reovirus to invade tree shrew,suggesting that tree shrew may be used as a new animal model of reovirus.

关 键 词：连接黏附分子A 基因克隆 呼肠孤病毒 树鼩 

分 类 号：Q95-33[生物学—动物学]