猪圆环病毒2型REP蛋白的原核表达及免疫原性鉴定
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摘要
[目的]开发和研制鉴别猪圆环病毒疫苗免疫和野毒感染的ELISA检测试剂盒。[方法]根据NCBI数据库录入的2型猪圆环病毒(porcine circovirus 2,PCV-2) REP蛋白序列(Gen Bank登录号为KX828581)设计引物,利用PCR扩增REP蛋白全长基,构建原核表达载体p ET-28a-rep,转化大肠杆菌BL21(DE3)感受态细胞,并诱导重组蛋白表达。[结果]经过SDS-PAGE分析,获得了大小约40 ku的目的蛋白,与理论大小相一致。重组蛋白经镍柱纯化后重组蛋白浓度为40μg/m L,纯度约95%。Western Blot及ELISA鉴定结果显示,该重组蛋白具有良好的免疫原性和反应原性。[结论]鉴于该重组蛋白具有良好的免疫原性及反应原性,该重组蛋白可为开发区分猪圆环病毒2型疫苗免疫和野毒感染的检测试剂盒奠定基础。
[Objective]To develop and produce Elisa kits for distinguishing vaccine immunization of porcine circovirus and wild virus infection.[Method]Based on the REP full length gene sequence of PCV-2( GenBank ID: KX828581),a pair of specific primers were designed for the amplification of the REP gene sequence. The prokaryotic expression vector p ET-28 a-rep was constructed,E.coli BL21( DE3) competent cell was transformed and the expression of recombinant protein was induced. [Result]Through SDS-PAGE,target protein with the size of about40 ku was obtained,which was accordant with the theoretical size. The recombinant protein was purified by using nickel column,and the purified recombinant protein concentration was 40 μg/m L,the purity was about 95%. Western Blot and ELISA results showed that the recombinant protein had good immunogenicity and reactivity. [Conclusion]The recombinant protein could lay the foundation for developing ELISA kits for distinguishing vaccine immunization of PCV-2 and wild virus infections.
[Objective]To develop and produce Elisa kits for distinguishing vaccine immunization of porcine circovirus and wild virus infection.[Method]Based on the REP full length gene sequence of PCV-2( GenBank ID: KX828581),a pair of specific primers were designed for the amplification of the REP gene sequence. The prokaryotic expression vector p ET-28 a-rep was constructed,E.coli BL21( DE3) competent cell was transformed and the expression of recombinant protein was induced. [Result]Through SDS-PAGE,target protein with the size of about40 ku was obtained,which was accordant with the theoretical size. The recombinant protein was purified by using nickel column,and the purified recombinant protein concentration was 40 μg/m L,the purity was about 95%. Western Blot and ELISA results showed that the recombinant protein had good immunogenicity and reactivity. [Conclusion]The recombinant protein could lay the foundation for developing ELISA kits for distinguishing vaccine immunization of PCV-2 and wild virus infections.
引文
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[8]崔尚金,全滟平,李曦,等.猪圆环病毒多重PCR诊断方法的建立[J].中国预防兽医学报,2006,28(5):581-584.
[9]崔尚金,全滟平,李曦,等.猪圆环病毒间接免疫荧光方法的建立[J].中国预防兽医学报,2007,29(1):63-66.
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[11]PENG Z Y,MA T,PANG D X,et al.Expression,purification and antibody preparation of PCV2 Rep and ORF3 proteins[J].Int J Biol Macromol,2016,86:277-281.
[2]JIANG C G,WANG G,TU Y B,et al.Genetic analysis of porcine circovirus type 2 in China[J].Archives of virology,2017,162(9):2715-2726.
[3]刘金鑫,孙孟娇,陈磊,等.猪圆环病毒研究进展[J].动物医学进展,2012,33(2):94-97.
[4]TISCHER I,GELDERBLOM H,VETTERMANN W,et al.A very small porcine virus with circular single-stranded DNA[J].Nature,1982,295(5844):64-66.
[5]CHEUNG A K.Identification of an octanucleotide motif sequence essential for viral protein,DNA,and progeny virus biosynthesis at the origin of DNA replication of porcine circovirus type 2[J].Virology,2004,324(1):28-36.
[6]MANKERTZ A,CALISKAN R,HATTERMANN K,et al.Molecular biology of Porcine circovirus:Analyses of gene expression and viral replication[J].Veterinary microbiology,2004,98(2):81-88.
[7]马萍,汤德元.猪圆环病毒基因及其疫苗的研究进展[J].猪业科学,2009,26(2):76-80.
[8]崔尚金,全滟平,李曦,等.猪圆环病毒多重PCR诊断方法的建立[J].中国预防兽医学报,2006,28(5):581-584.
[9]崔尚金,全滟平,李曦,等.猪圆环病毒间接免疫荧光方法的建立[J].中国预防兽医学报,2007,29(1):63-66.
[10]董林.猪圆环病毒2型诊断试剂盒的研制与应用[D].长春:吉林大学,2014.
[11]PENG Z Y,MA T,PANG D X,et al.Expression,purification and antibody preparation of PCV2 Rep and ORF3 proteins[J].Int J Biol Macromol,2016,86:277-281.