猪圆环病毒2型ORF1、ORF2和ORF3原核表达和多克隆抗体的制备  

Prokaryotic expression and polyclonal antibodies preparation of ORF1,ORF2 and ORF3 of porcine circovirus type 2

作　　者：陈禹[1] 杜谦[1] 霍瑞超[1] 王莉莉[1] 童德文[1] 黄勇[1] 

机构地区：[1]西北农林科技大学动物医学学院,陕西杨凌712100

出　　处：《中国兽医学报》2015年第5期686-691,共6页Chinese Journal of Veterinary Science

基　　金：西北农林科技大学基本科研业务资助项目(ZD2013009;Z111021103);2011教育部博士点基金资助项目(20110204110014)

摘　　要：猪圆环病毒2型(porcine Circovirus type 2,PCV2)感染给养猪业造成巨大的经济损失,而PCV2ORF1、ORF2和ORF3与PCV2的复制、免疫原性及致病机理密切相关。本研究制备PCV2ORF1、ORF2和ORF3多克隆抗体,为进一步研究PCV2ORF1、ORF2和ORF3的功能提供材料。依据PCV2全基因组序列,分别设计针对PCV2ORF1、ORF2和ORF3编码基因的特异性引物,PCR扩增后,克隆入原核表达载体pET-32a的BamHⅠ/NcoⅠ和HindⅢ位点之间,经PCR、双酶切和测序鉴定正确的重组原核表达载体分别命名为pET-ORF1、pET-ORF2和pET-ORF3。重组质粒分别转化E.coli BL21菌株,1 mmol/L IPTG诱导表达ORF1、ORF2和ORF3重组蛋白,SDS-PAGE测定结果表明其相对分子质量分别约为52 000、39 000和29 000。大量诱导重组蛋白表达后,回收目的蛋白条带、匀浆后免疫新西兰大白兔,制备兔抗PCV2ORF1、ORF2和ORF3重组蛋白血清。兔抗血清纯化后,Western blot检测抗血清与重组蛋白的特异性结合情况及其效价,证明所制备多克隆抗体均具有较强的特异性,效价均高于1∶10 000。Porcine circovirus type 2(PCV2)has caused huge economic losses to the pig industry.PCV2 open reading frames(ORF1,ORF2 and ORF3)are closely related to replication,immunogenicity and pathogenesis of PCV2.To further study the function of PCV2ORF1,ORF2 and ORF3,polyclonal antibodies were prepared in this work.Specific primers for the genes of ORF1,ORF2 and ORF3were designed and synthesized according to PCV2 genome sequences.The ORF1,ORF2 and ORF3genes were amplified by PCR and cloned to the expression vector pET-32 aat the sites between BamHⅠ/NcoⅠ and HindⅢ,identified by specific PCR,enzyme digestion and sequencing recombination plasmids were named pET-ORF1,pET-ORF2 and pETORF3,respectively.These recombinant plasmids were transfected into E.coli BL21 and the cells were treated by 1mmol/L IPTG to express the interest proteins of ORF1,ORF2 and ORF3.SDS-PAGE analysis showed the molecular weight of ORF1,ORF2 and ORF3encoded proteins were about 52 000,39 000 and 29000,respectively.The target proteins were separated and collected,then immunized New Zealand White Rabbit to acquire the antibodies against ORF1,ORF2 and ORF3.After purification of these antibodies via nonspecific adsorption,western blot analysis showed that all of these three antibodies were specific for their target proteins and exhibited higher titer(exceed 1∶10000).

关 键 词：PCV2 ORF1 ORF2 ORF3 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]