利用番鸭整合素α4亚基多克隆抗体对MDRV感染雏番鸭淋巴细胞归巢的研究  

Investigation of lymphocyte homing in MDRV-infected Muscovy ducklings using polyclone antibody specifically recognizing duck integrin α4

作　　者：刘珍妮 李明慧 骆钰 刘秉珲 黄诗杭 吴宝成[1,2] 黄一帆 吴异健[1,2] LIU Zhen-ni;LI Ming-hui;LUO Yu;LIU Bing-hui;HUANG Shi-hang;WU Bao-cheng;HUANG Yi-fan;WU Yi-jian(Fujian Agriculture and Forestry University,Fuzhou 350002,China;Fujian Key Laoratory of Traditonal Chinese Veterinary Medicine and Animal Health,Fuzhou 350002,China)

机构地区：[1]福建农林大学动物科学学院,福建福州350002 [2]福建省兽医中药与动物保健重点实验室,福建福州350002

出　　处：《中国预防兽医学报》2019年第8期847-852,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：福建省自然科学基金项目(2017J01597)

摘　　要：为研究番鸭呼肠孤病毒(MDRV)感染鸭后肠淋巴细胞归巢中的细胞定位,本研究通过RT-PCR方法扩增番鸭肠道α4编码基因,构建重组表达质粒p ET-His-α4,将其转化E.coli BL21 (DE3)表达了r His-α4,以其为抗原免疫福建黄兔,制备了抗r His-α4多克隆抗体,该多克隆抗体的间接ELISA和琼扩效价分别为1∶52 000和1∶32。利用制备的多抗对MDRV感染的番鸭淋巴细胞进行western blot和间接免疫荧光检测,结果显示体外淋巴细胞在感染MDRV后,α4蛋白的表达量呈增长趋势;雏番鸭感染MDRV后盲肠的α4+淋巴细胞增加的同时CFSE+淋巴细胞也显著增加,表明MDRV感染后诱导淋巴细胞大量表达了α4整合素,导致淋巴细胞向肠道黏膜组织归巢。本研究制备的番鸭r His-α4兔源多克隆抗体可以用于番鸭整合素α4的检测,同时也为进一步阐明MDRV感染诱导淋巴细胞归巢的分子机制研究奠定了基础。To investigate the process of intestinal lymphocyte homing in ducks upon MDRVinfection, polyclone antibody targeting duck integrin α4 was prepared in present study. The DNA fragment encoding integrin α4 of Muscovy duck was amplified by RT-PCR and inserted into pET-32 a(+) to construct the prokaryotic expression plasmid pET-His-α4. The resultant recombinant plasmid was transformed into BL21(DE3) to expressrHis-α4. The anti-integrin α4 polyclonal antibody was prepared in rabbits using r His-α4(1.0 mg) as antigen. The highest titers of this antibody for indirect ELISA and AGARcan achieve 1 ∶512 000 and 1∶32 respectively. Using this polyclonal antibody, western blot assay and indirect immunofluorescence histochemistry assay of samples collected from uninfected and MDRV-infected Muscovy ducklingswere performed. The results showed that MDRV infection promoted the expression of α4 protein in extracorporeal lymphocyte. Besides, both the numbers of α4+lymphocytes and CFSE+lymphocytes in the cecum of Muscovy ducklings were increased by MDRV infectionsignificantly, indicating that a large amount ofintegrin α4 was induced by MDRV infection in lymphocytes, which maylead to lymphocytes homing in intestinal mucosal tissue.The present study developed a polyclonal antibody targeting duck integrin α4, which can be used for the detection of lymphocyte homing in duck, and lays afoundation for further study of the biological function of duck integrin α4 protein.

关 键 词：番鸭整合素α4 原核表达 多克隆抗体 番鸭呼肠孤病毒 淋巴细胞归巢 

分 类 号：S852.65[农业科学—基础兽医学]