猪FMDV受体整联蛋白β1亚基LBD多克隆抗体的制备和初步应用  

Preparation and preliminary application of the polyclonal antibody against ligand binding domain of procine integrin β1 subunit of foot and mouth disease virus

作　　者：高闪电[1] 常惠芸[1] 独军政[1] 王景锋[1] 宋帅[1] 谢庆阁[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,国家口蹄疫参考实验室,家畜疫病病原生物学国家重点实验室,农业部畜禽病毒学重点开放实验室,兰州730046

出　　处：《中国人兽共患病学报》2008年第11期999-1001,1005,共4页Chinese Journal of Zoonoses

基　　金：国家重点基础研究发展计划(973)项目(NO.2005CB523201);国家支撑计划资助项目(2006BAD06A14;6BAD06A03)

摘　　要：目的表达口蹄疫病毒受体猪源整联蛋白β1亚基配体结合域β1LBD,制备兔抗β1LBD多克隆抗体检测β1p-CHOK1细胞β1亚基的表达。方法利用PCR方法扩增整联蛋白β1LBD基因片段,经BamHⅠ和XhoⅠ进行双酶切后与pGEX-4T-1原核表达载体相连,构建的pGEX-β1LBD重组表达质粒,转化大肠杆菌BL21(DE3)菌株,经IPTG诱导表达,SDS-PAGE鉴定融合蛋白的表达并提取包涵体,纯化目的蛋白。然后用纯化的蛋白免疫新西兰大耳白兔制备其多克隆抗体,以ELISA和Western blot方法分别鉴定抗体的效价和特异性,并用制备的抗体以间接免疫荧光抗体法对β1p-CHOK1细胞中β1亚基的表达进行检测。结果SDS-PAGE电泳分析显示阳性菌经诱导后表达一Mr约为42000的GST-β1LBD融合蛋白,与预期结果相符。目的蛋白纯化后,在电泳图片上显示较为清晰的单一条带,用纯化GST-β1LBD免疫新西兰大耳白兔制备的多克隆抗体效价达1∶12800以上,具有较高的特异性,间接免疫荧光显示β1p-CHOK1细胞β1亚基表达于其细胞表面。结论制备了具有高亲和性和特异性的兔抗猪源整联蛋白β1LBD多克隆抗体,为深入研究猪源整联蛋白β1在口蹄疫病毒感染过程中的作用了奠定基础。In order to express the ligand binding domain of porcine integrin β1 subunit（β1LBD） as receptor of foot and mouth disease virus（FMDV）, the polyclonal antibody against β1LBD was prepared in rabbits and used to detect the expression of the integrin β1 subunit in β1p-CHOK1 cells. The gene fragment coding β1LBD was amplified with PCR and doubly digested with BamH1 and Xhol, then cloned into expression vector pGEX-4T-1 to obtain the recombinant plasmid pGEX-β1LBD. This recombinant plasmid was then transformed to E. coli BL21（DE3） and induced by IPTG from which the fusion protein was identified by SDS-PAGE. Later on the polyclonal antibody was prepared by immunization of New Zealand rabbits with purified fusion protein used to detect the expression of β1LBD, and was characterized by ELISA and Western blot assay. The expression of integrin β1 in β1-transfeeted CHOK1 cells was detected by indirect immunofluorescence assay（IFA）. As demonstrated by SDS-PAGE, the fusion protein GST-β1LBD was expressed with the expected molecular weight of 42 000 and a single clear band of the GST-β1LBD fusion protein appeared in SDS-PAGE gel. The titer of the polyclonal antibody was over 1 ： 12 800 and this antibody showed obvious specificity. The integrin β1subunit was found to be expressed mainly on the surface of integrin β1ptransfected CHOKI cells as demonstrated by IFA. It is apparent that the rabbit anti-procine β1LBD poloyclonal antibody with high affinity and specificity was successfully prepared ,which will lay a foundation for further investigation on the role of integrin β1 in FMDV infection.

关 键 词：口蹄疫病毒 受体 整联蛋白β1亚基 配体结合域 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]