猪PRRSV受体唾液酸黏附素多克隆抗体的制备  

Preparation of polyclonal antibody against PRRSV receptor sialoadhesin

作　　者：李振[1] 仲飞[1] 李秀锦[2] 王幸兴[1] 韩冬梅[1] 张峰[1] 潘红丽[1] 王璐[1] 孙岩[1] 贾启恒[1] 

机构地区：[1]河北农业大学动物医学院农业部动物疫病病原生物学华北科学观测实验站河北省兽用生物制品工程技术研究中心,河北保定071001 [2]燕山大学环境与化学工程学院,河北秦皇岛066004

出　　处：《中国兽医学报》2013年第2期181-186,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31140093);河北省自然科学基金资助项目(C2008000244)

摘　　要：唾液酸黏附素(sialoadhesin,Sn)是猪呼吸与繁殖综合征病毒(PRRSV)的主要受体,研究该受体在PRRSV感染过程中的作用依赖于特异的抗体。为制备Sn的抗体,首先依据通过DNAstar软件分析的抗原指数,确定编码114个氨基酸的Sn抗原表位基因(Sn114),然后依据大肠杆菌偏爱的密码子对该基因进行密码子优化,并由人工合成该基因。将合成的基因克隆到pET-32a(+)质粒中构建成与硫氧还蛋白(Trx)和6×His融合的Sn114基因原核表达载体,将表达载体转化BL21(DE3)大肠杆菌,在IPTG的诱导下使其表达。利用Ni-NTA琼脂糖分离纯化表达的Sn114融合蛋白,再用Sn114融合蛋白免疫家兔,通过ELISA方法检测免疫后家兔血清的抗体滴度,并通过Westernblot和细胞结合试验检测抗体的特异性。结果表明,本试验构建的Sn114表达载体能够介导Sn114融合基因在大肠杆菌中进行高效表达,表达量为63mg/L;用纯化后的Sn114蛋白免疫家兔,免疫后34d抗体滴度为1∶10 240。抗体特异性检测结果表明,所制备的Sn抗体能与猪肺泡巨噬细胞的Sn抗原进行特异性免疫反应。SialoadheSin（Sn） is a primary receptor of porcine reproductive and respiratory syndrome virus（PRRSV）. The specific antibody against Sn is required for investigation of Sn functions during PRRSV infection. To prepare Sn antibody, an epitope gene of Sn, coding 114 amino acids （Sn114） was determined based on the antigenic analysis of complete Sn sequence by DNAstar software. The Snll4 epitope gene sequence was codon-optimized according to E. coli preferred codons and synthesized by a company. The codon-optimized Sn114 gene was cloned into pET-32a （＋） plasmid to construct the prokaryotic expression vector of Snll4 gene fused with thioredoxin and His-tag. The expression vector was transformed into E. coli BL21（DE3） to express Snll4 protein with IPTG induction. The Sn114 fusion protein was purified with Ni-NTA agarose beads. The rabbits were immunized with the Sn114 fusion protein, and the antibody titer was measured by ELISA. The result showed that the Sn114 fusion protein was highly expressed in E. coli at,about 63 mg/L. The serum antibody titer reached 1 ： 10 240 at 34 d after immunization. The macrophage biding assay indicated that the antibody had the ability to specifically detect Sn antigen on porcine alveolar macrophages.

关 键 词：唾液酸黏附素 密码子优化 多克隆抗体 PRRSV 

分 类 号：S852.65[农业科学—基础兽医学]