摘要
根据GenBank发表的猪圆环病毒2型(PCV2)基因序列设计并合成1对特异性引物,进行PCR扩增,从PCV2突变株中扩增出PCV2 Rep基因,对PCR扩增产物清洁后进行双酶切,连接到pEGFP-C1载体上,转化到大肠杆菌DHSα感受态细胞中,经PCR筛选和测序后鉴定出正确的重组基因,完成真核表达载体的构建。将重组质粒转染到PK15细胞中,收取病毒液抽提DNA,PCR检测到Rep基因,证明转染是成功的。进行间接免疫荧光试验,可以检测Rep基因在PK15细胞中的表达,试验结果很好的验证了Rep基因的免疫原性。这为进一步研究PCV2的生物学活性及PCV2新型疫苗奠定了坚实的基础。
A pair of specific primers was designed and synthesized according to the porcine circovirus type2（PCV2） gene sequence published in the GenBank,the PCV2 Rep gene was amplified by PCR from mutant strains of PCV2,and after cleaning and double digests the amplified products were connected to the pEGFP-Cl vector and transformed into E.coli DH5α competent cells.The correct recombinant gene was identified by PCR screening and sequencing,indicating the construction of eukaryotic expression vector was finished.The recombinant plasmid was transfected into PK15 cells,and then the virus liquid was collected to extract the DNA.The Rep gene detected by PCR proved that the transfection was successful.The experimental results well verified the immunogenicity of Rep gene in PK15 cells,thus laying a solid foundation for further study of the biological activities and new vaccine of PCV2.
出处
《上海农业学报》
CSCD
2016年第1期6-9,共4页
Acta Agriculturae Shanghai
基金
上海市科学技术委员会重点科技攻关项目(12391901900)
关键词
猪圆环病毒2型
Rep基因
真核表达载体
间接免疫荧光
Porcine circovirus type 2
Rep gene
Eukaryotic expression vector
Indirect immunofluorescence
