猪圆环病毒2型Rep和Rep’蛋白免疫原性及其抗体中和活性的测定

Determination of immunogenicity and neutralizing activity of the Rep and Rep' proteins derived from porcine circovirus type 2

为了构建表达PCV2Rep和Rep’蛋白的重组杆状病毒,采用高保真PCR法从病毒感染细胞中扩增Rep和Rep’蛋白基因,并克隆到杆状病毒转移载体(pBlueBac4.5/V5-His-TOPO)。将重组质粒与线性化杆状病毒DNA共转染昆虫细胞(Sf21),经同源重组和蚀斑筛选,获得2株高效表达PCV2Rep和Rep’蛋白的重组杆状病毒。经3次克隆的重组毒株的毒价达3.0×107 PFU/mL和7.0×107 PFU/mL。蛋白表达产物分析表明,PCV2Rep和Rep’蛋白分子质量为36.0ku和20.4ku,分别占总蛋白含量的6.2%和10.1%。免疫活性分析表明,这2种重组蛋白均可与PCV2特异性抗体产生反应,具有良好的反应原性。用Ni-NTA亲和层析法纯化这2种重组蛋白,纯度分别为92.5%和93.6%。用纯化的2种蛋白免疫小鼠,经间接ELISA检测,血清抗体效价高达1∶51 200。以抗这2种蛋白的抗体进行的IPMA试验表明,它们均能特异性识别PCV2感染细胞中的Rep和Rep’蛋白抗原。病毒血清中和试验证实,抗Rep和Rep’蛋白抗体均无中和病毒活性。本研究表达的重组PCV2Rep和Rep’蛋白,为探讨该病毒复制机制奠定了基础,也为PCV2Cap亚单位疫苗与自然野毒感染间的鉴别诊断开辟了新途径。

To construct the recombinant baculovirus system for expressing the replicase（Rep） and Rep＇ proteins,their genes were amplified using high fidelity PCR and cloned into a transfer vector of pBlueBac4.5/V5-His-TOPO.The recombinant plasmid with a mixture of linearized baculovirus DNA were co-transfected into the insect cells（Sf21）.After three circles of plaque clone,two recombinant baculoviruses highly expressing PCV2 Rep and Rep＇ proteins were obtained.The titers of two mast seed viruses reached to 3.0×10^7 PFU/mL and 7.0×10^7 PFU/mL,respectively.The molecular weight of the recombinant Rep and Rep＇ proteins were 36.0 ku and 20.1 ku and accounted for 6.2% and 10.1% in the total protein by SDS-PAGE analysis,respectively.The Rep and Rep＇ proteins had strong reaction with anti-PCV2 antibody in Western-blot analysis.The purity of the Rep and Rep＇ proteins were 92.5% and 93.6% after purification by Ni-NTA affinity chromatography.The immunogenicity of the Rep and Rep＇ were determined by immunization of BALB/c mice.The antibodies against the Rep and Rep＇ were evaluated by ELISA,and both antibody titers were more than 1∶51 200.Two kinds of antibodies were reacted with the PCV2 Rep and Rep＇ in the virus-infected cells,but there were not neutralizing activity observed in serum neutralization test.The recombinant PCV2 Rep and Rep＇ proteins provide the basis for further research on the virus replication mechanisms,and that would be useful for establishing diagnostic method to distinguish PCV2 Cap subunit vaccination with wild type virus infection.