猪圆环病毒3型Rep蛋白的表达与多克隆抗体制备

Prokaryotic expression of Rep protein of porcine circovirus type 3 and preparation of polyclonal antibodies against Rep protein

为研究猪圆环病毒3型(PCV3)Rep基因在大肠杆菌中表达产物的反应原性和免疫原性,本试验对Rep基因进行原核表达和纯化,鉴定表达产物与PCV3阳性血清的反应性;制备重组Rep蛋白的多克隆抗体,Western blot鉴定多克隆抗体与重组Rep蛋白的特异性识别能力,通过ELISA测定多克隆抗体的效价。结果显示,Rep在大肠杆菌中获得大量表达,且主要以包涵体形式存在,重组蛋白相对分子质量约为32000,经纯化后得到单一条带。表达的重组Rep蛋白可以与PCV3阳性血清发生反应。制备的大鼠抗Rep血清与表达的重组Rep蛋白发生特异性反应,且效价大于1∶32000。表明本试验表达的Rep具有良好的免疫原性和反应原性,为PCV3的ELISA诊断试剂盒研制提供依据。

In order to study the reactionogenicity and immunogenicity of expression product of porcine circovirus type 3(PCV3)Rep gene in Escherichia coli(E.coli),the recombinant Rep protein was expressed in E.coli BL21(DE3)and purified.It was identified whether the recombinant Rep protein could react to the PCV3positive serum.The polyclonal antibody against Rep protein was prepared,and its titer was detected by ELISA.It was determined whether the polyclonal antibody specifically recognize the recombinant Rep protein by Western blot.The results showed that the recombinant Rep protein could be expressed in E.coli BL21(DE3),mainly in inclusion body.Molecular weight of recombinant Rep protein was about 32000.The recombinant Rep protein had reactionogenicity to the PCV3-positive serum.There was4.9 c381.a clear singly target band of the purified Rep protein.The titer of the polyclonal antibody was higher than 1∶32000.The polyclonal antibody could specifically recognize the recombinant Rep protein.This study suggested that the recombinant Rep protein had good reactionogenicity and immunogenicity,which provided a foundation for developing the ELISA diagnostic kit.