猪PCV2 ORF2蛋白I-ELISA检测方法的建立及猪场血清抗体检测分析

Establishment of application I-ELISA method for pig PCV2 ORF2 protein

目的用猪PCV2 ORF2蛋白建立ELISA检测方法 ,有效区分猪圆环病毒1型(PCV1)和猪圆环病毒2型(PCV2),并进行大规模化样本检测。方法以猪PCV2 ORF2蛋白作为包被抗原,以抗PCV2阳性血清为一抗,羊抗猪酶标物为二抗,通过预试验确定诱导温度、诱导时间、摇动转速、IPTG浓度以及反应板种类等参数,从而建立间接酶联免疫吸附方法(I-ELISA)以检测抗PCV2猪血清抗体滴度。结果采用本研究建立的ELISA方法测定山东省内4个猪场送检的316份猪血清,检出PCV2阳性率处于较高水平,表明猪群携带病毒比例很高。结论本试验建立的间接ELISA检测方法敏感度高、特异性强,可用于临床PCV2血清抗体的检测,为ELISA诊断试剂盒的研制开发奠定了基础,为建立规模化猪场PCV2感染的快速诊断方法提供了科学依据。

This study desgined to establish an ELISA method by using PCV2 ORF2 protein, which can distinguish the difference between PCV1 and PCV2 virus, and can be applied for large-scale sample detection. The truncated ORF2 gene of PCV2 was cloned into the prokaryotic expression vector p GEX-4T-1 and expressed in the competent cells E. coli BL21（DE3） induced by the IPTG. Taking the expressed PCV2 ORF2 protien as envelope protein, an indirect ELISA method was established through pre-testing several parameters（temperature, IPTG concentration, inducing time, et al）. Total of 316 sera submitted from four pig farms of Shandong province were tested by the referred indirect ELISA, and the data showed that the seroprevalence specific to PCV2 was in superior high degree. Teaken togather, the expected indirect ELISA is established successfully, which is high sensitivity,strong specificity, and can be used for clinical PCV2 serum antibody detection. And our study will also lay a foundation for development of ELISA diagnostic kit used for large-scale rapid diagnosis of PCV2.