中国兽医科学2017，Vol.47Issue(4)：427-434,8.DOI:10.16656/j.issn.1673-4696.2017.04.004
基于ORF3蛋白的PCV2间接ELISA诊断方法的建立
Establishment of an indirect ELISA for the detection of antibodies against PCV2 based on recombinant ORF3 protein
摘要
Abstract
ORF3 protein is an important non-structural protein of porcine circovirus type 2 (PCV2).A pair of primers were designed based on 0RF3 gene of PCV2 retrieved from GenBank,and used to amplify the ORF3 geneby PCR.Thepurified PCR production was recombined into pET-32a (+).In addition,the recombinant plasmid was transferred into BL21(DE3)competent cells.The recombinant protein expression conditions were selected and detected by SDS-PAGE electrophoresis.The antigenicity of the recombinat protein was identified by Western-blot.The recombinant protein was purified with ProteinIso Ni-NTA resin.The antidoy of ORF3 was prepared and the antibody titer was detected by the indirect ELISA.In result,the recombinant plasmid pET-32a(+)-ORF3 was constructed successfully,and was identified by PCR,enzymedigestion and sequencing.Optimal induction conditions for protein expression were 37℃,8 h and 1 mmol/L IPTG.Identification by Western-blot showed that the recombinant protein had a good immunogenicity.Ni-NTA affinity chromatography results showed that the best elution concentration of imidazole was 280mmol/L.The optimal concentration of coating antigen was 0.03 mg/mL.The serum sample was diluted by 1∶100 and HRPconjugated goat anti-pig IgG was diluted with 1∶4 000.The intra-batch and inter-batches test showed that it had good repeatability.It is concluded that the iELISA can be used as an indentification method to tell immuization and natural infection.关键词
猪圆环病毒2型/ORF3/原核表达/间接ELISAKey words
porcine circovirus virus 2/ORF3/prokaryotic expression/indirect ELISA分类
农业科技引用本文复制引用
李春燕,刘梦茜,陈仕怡,袁晓琴,星东,庄育彬,王全溪..基于ORF3蛋白的PCV2间接ELISA诊断方法的建立[J].中国兽医科学,2017,47(4):427-434,8.基金项目
福建农林大学科学发展基金项目(KF2015094) （KF2015094）
