猪FMDV受体β3亚基配体结合域的基因克隆及其多克隆抗体制备

猪FMDV受体B3亚基配体结合域的基因克隆及其多克隆抗体制备

（作者：_________ 单位：___________ 邮编：___________ ）

作者：独军政，高闪电，常惠芸，丛国正，林彤，邵军军, 刘湘涛，才学鹏

【摘要】目的：克隆猪FMDV受体B3亚基的配体结合域（LBD）基因，利用原核细胞表达B 3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B 3亚基的LBD基因，将其与pGEM T-easy载体相连构建pGEM/ P3LBD,经BamH /Xho I酶切后回收3LBD片段，与同样酶切处理的原核表达载体pGEX 4T-1相连，构建原核表达质粒pGEX/ （33LBD,将其转化感受态细胞BL21（DE3）,以IPTG诱导表达

P3LBD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B3亚基的LBD基因含有507个核苷酸，编码169个氨基酸，猪B3 LBD基因与牛、人、黑猩猩、猕猴、马、犬、挪威大鼠、家鼠、和鸡的B3 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B3亚基LBD基因同源性较高，与鸡的同源性较低。实现了重组猪B 3 LBD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44 000,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B3 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B 3亚基在FMDV感染过程中的作用机制奠定了基础。

【关键词】FMDV病毒受体猪(33LBD基因原核表达多克隆抗体[Abstract ] AIM: To clone and express the ligand binding domain (LBD) cDNA of porcine integrin [33 as foot-and-mouth disease virus (FMDV) receptor and prepare its polycl onal an tibody. METHODS: The LBD cDNA of porci ne [33 was obta in ed from the lung tissue of pig infected with FMDV by RT-PCR, and the recomb inant plasmid pGEM/ 3 3LBD was con structed. After digested with BamH /Xho [the [33LBD fragme nt was subcl oned into prokaryotic expressi on vector pGEX 4T-1. The recomb inant expression

plasmid pGEX/ 3 3LBD was constructed and tran sformed into E.coli BL21(DE3). The recomb inant porc ine 3 3LBD prote in was expressed after IPTG in ducti on 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 B3LBD encoded a polypeptide of 169 amino acids. The similarity of nucleotide sequenee [3 3LBD between pigs and cattle, human being,

chimpa nzees, rhesus mon keys, 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 [3 3LBD gene of mammals

exhibited high seque nee homology. The recomb inant [33LBD protein was expressed efficiently as inclusion body after IPTG induction and was approximately 44 000. The titer of the polyclonal an tibody agai nst the purified 33LBD protein was about 1 12800 by ELISA. CONCLUSION: The gene cloning and expression of 3 3LBD and the preparation of its polyclonal antibody lay a foun dati on for further research in to the in teracti on of FMDV with [33 subunit of porcine integrin.

[Keywords ] FMDV receptor; ligand binding domain cDNA of porc ine [33; prokaryotic expressi on; polycl onal an tibody

病毒感染宿主细胞的第一个步骤是病毒与受体结合，在受体的介导下病毒粒子才能进入细胞内。已经发现的口蹄疫病毒 (foot-and-mouth disease, FMDV )受体分为H类：整联蛋白 (integrin )和硫酸乙酰肝素(heparan sulfate proteoglycans, HSPG ) [1]。口蹄疫田间分离毒株仅利用整联蛋白作为受体，而口蹄疫细胞适应毒株则除了利用整联蛋白