摘要
为了使猪圆环病毒2b型(PCV2b)Cap蛋白在猪圆环病毒病等疾病的诊断和防制方面取得更有效的应用,本试验将编码PCV2bCap蛋白的ORF2基因克隆到原核表达载体pET-32a(+)中,并构建pET-32a-ORF2重组质粒,重组质粒转化大肠杆菌BL21受体菌后,通过IPTG诱导表达重组蛋白,将重组蛋白用镍柱进行纯化并免疫大白兔,制备兔抗PCV2bCap蛋白的多克隆抗体。利用ELISA、Western blotting、间接免疫荧光及病毒交叉反应等试验对制备的多克隆抗体进行生物学特性检测。ELISA检测结果显示,该抗体效价可达到1∶216;Western blotting检测结果显示,该多克隆抗体可与PCV2b产生特异性的反应条带,说明其具有较好的反应活性;间接免疫荧光试验结果显示,该多克隆抗体能识别感染PK-15细胞中的PCV2b,说明该多克隆抗体具有鉴别诊断PCV2b的能力;病毒交叉试验结果显示,该多克隆抗体能与PCV2b反应,而不与猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪轮状病毒(PRoV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)等猪源病毒发生交叉反应,表明该多克隆抗体具有高度特异性。本试验制备的抗PCV2bCap蛋白多克隆抗体为PCV2b病原特性研究及该病的临床检测奠定基础。
In order to build more effective use of porcine eircovirus type 2b （PCV2b） Cap protein on the diagnosis and control of diseases like porcine circovirus disease, the ORF2 gene which enco- ding porcine circovirus type 2b Cap protein was cloned into the prokaryotic expression vector pET-32a （＋）, constructing a recombinant plasmid pET-32a-ORF2,then the recombinant plasmid was transformed into E. coli BL21 and induced by IPTG to express recombinant protein, the re- combinant protein was purified with nickel column. Then polyclonal antibody （PcAb） was pre- pared by immunization of rabbit with purified protein. ELISA, Western blotting,indirect immuno- fluorescence assay and the specificity experiments were used to detect the biological characteristics of the polyclonal antibody. The titer of polyclonal antibody was about 1 ： 2^16 detected by ELISA.Western blotting result confirmed that polyclonal antibody could react with PCV2b specially. In- direct immunofluorescence assay showed that polyclonal antibody was able to detect PCV2b in PK-15 cells,this result suggested that the polyclonal antibody had the ability to differential diag- nosis of PCV2b. The specificity experiment showed that the polyclonal antibody only reacted with PCV2b,and did not react with PEDV,TGEV,PRRSV,PRoV,PRV and PPV. The polyclonal anti- body against PCV2b prepared in this study provided a powerful tool for the study of etiological characteristics and clinic diagnosis of this disease.
出处
《中国畜牧兽医》
CAS
北大核心
2015年第5期1048-1055,共8页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31200122
31270187)
教育部科学研究重点项目(212038)
哈尔滨市科技局"优秀学科带头人"研究专项资金(2012RFXXN009)
关键词
猪圆环病毒2b型
CAP蛋白
原核表达
多克隆抗体
生物学特性
porcine circovirus type 2b
Cap protein
prokaryotic expression
polyclonal antibody
biological characteristics
作者简介
袁庆（1987-），男，四川绵阳人，硕士，研究方向：病原微生物与免疫，E—mail：yuanqing4216@163．com
通信作者：李广兴（1968-）。男，黑龙江哈尔滨人，博士，教授，博士生导师，主要从事动物病理学研究，E-mail：ligx@neau．edu．cn；Tel：0451-55190723
