摘要
目的:克隆猪FMDV受体β3亚基的配体结合域(LBD)基因,利用原核细胞表达β3亚基的LBD片段,纯化目的蛋白后制备兔多克隆抗体。方法:利用RT-PCR方法从FMDV实验感染猪的肺组织中克隆β3亚基的LBD基因,将其与pGEMT-easy载体相连构建pGEM/β3 LBD,经BamHⅠ/XhoⅠ酶切后回收β3LBD片段,与同样酶切处理的原核表达载体pGEX4T-1相连,构建原核表达质粒pGEX/β3 LBD,将其转化感受态细胞BL21(DE3),以IPTG诱导表达β3LBD蛋白。纯化目的蛋白后免疫新西兰白兔制备多克隆抗体。通过ELISA和Western blot鉴定血清效价和特异性。结果:猪β3亚基的LBD基因含有507个核苷酸,编码169个氨基酸,猪β3 LBD基因与牛、人、黑猩猩、猕猴、马、犬、挪威大鼠、家鼠、和鸡的β3 LBD基因核苷酸序列同源性分别为90·3%、92·3%、92·1%、91·3%、90·5%、90·3%、87·8%、85·2%、79·5%。哺乳动物的β3亚基LBD基因同源性较高,与鸡的同源性较低。实现了重组猪β3LBD蛋白在大肠杆菌中的高效表达,SDS-PAGE显示其相对分子质量(Mr)约为44000,制备的兔多克隆抗体效价达1∶12800以上。结论:实现了猪FMDV受体β3 LBD的基因克隆、原核表达和多克隆抗体的制备,为深入研究受体β3亚基在FMDV感染过程中的作用机制奠定了基础。
AIM： To clone and express the ligand binding domain （LBD） cDNA of porcine integrin β3 as foot-andmouth disease virus （FMDV） receptor and prepare its polyclonal antibody. METHODS： The LBD cDNA of porcine was obtained from the lung tissue of pig infected with FMDV by RT-PCR, and the recombinant plasmid pGEM/β3LBD was constructed. After digested with BamH I/Xho I, the β3LBD fragment was subcloned into prokaryotic expression vector pGEX 4T-1. The recombinant expression plasmid pGEX/β3LBD was constructed and transformed into E. coli BL21（ DE3 ）. The recombinant porcine β3LBD protein was expressed after IPTG induction and purified from total protein of BL21 （DE3）. The rabbits from New Zealand were immunized with the purified fusion protein to prepare polyclonal antibody, which was identified by Western blot and ELISA. RESULTS： The 507 bp cDNA of porcine β3LBD encoded a polypeptide of 169 amino acids. The similarity of nucleoUde sequence β3LBD between pigs and cattle, human being, chimpanzees, rhesus monkeys, horses, dogs, Norway rats, mice, chickens was 90.3%, 92.3%, 92.1%, 91.3%, 90.5%, 90.3%, 87.8%, 85.2%, 79.5%, respectively. The β3LBD gene of mammals exhibited high sequence homolo-gy, The recombinant β3LBD protein was expressed efficiently as inclusion body after IPTG induction and was approximately 44 000. The titer of the polyclonal antibody against the purified β3LBD protein was about 1 ： 12 800 by ELISA. CONCLUSION： The gene cloning and expression of 133 LBD and the preparation of its polyclonal antibody lay a foundation for further research into the interaction of FMDV with β3 subunit of porcine integrin.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2008年第4期358-361,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家支撑计划资助项目(2006BAD06A03)
国家高技术研究发展计划(863)资助项目(2006AA10A204)
关键词
FMDV病毒受体
猪β3LBD基因
原核表达
多克隆抗体
FMDV receptor
ligand binding domain cDNAof porcine β3
prokaryotic expression
poly-clonal antibody
