【摘要】 猪圆环病毒2型(PCV2)基因组包含2个开放阅读框架(ORFs),其中ORF1编码病毒复制相关蛋白(Rep),ORF2编码病毒衣壳蛋白(Cap)。为了在昆虫细胞表达Cap蛋白,本研究采用PCR扩增PCV2-ORF2编码基因,将PCR产物插入到昆虫杆状病毒转移载体上,经酶切反应及DNA序列分析得到验证。重组质粒与昆虫杆状病毒线性基因组混合,转染到昆虫细胞(Sf-21)进行基因重组,经3次病毒蚀斑克隆,获得高效表达Cap蛋白的重组杆状病毒,毒价可达1.28×108pfu/mL。采用SDS-PAGE凝胶电泳分析表明,重组Cap融合蛋白分子量为32.8 ku,占总蛋白含量的17.2%。免疫印迹试验分析表明,重组Cap蛋白与PCV2阳性血清产生特异性反应,证明该重组蛋白具有良好的免疫活性反应。本研究为进一步进行该病毒分子诊断、亚单位疫苗以及分子生物学等研究奠定了基础。更多还原

【Abstract】 Porcine circovirus type 2(PCV2) genome contains two major open reading frames(ORFs),ORF1 encoding the viral replication-associated protein(Rep) and ORF2 encoding the viral capsid protein(Cap).To express the Cap protein in insect cells,PCV2-ORF2 gene was amplified by PCR and inserted into a baculovirus transfer plasmid vector.The recombinant plasmid was confirmed using restriction enzymes analysis and DNA sequencing.The insect cells(Sf-21) were transfected with a mixture of linearized baculovirus DNA and the recombinant plasmid.A titre of 1.28×10~8 pfu/ml recombinant baculovirus expressing the Cap protein was obtained latter after three circles of plaque cloning.The recombinant fusion capsid protein with a molecular weight of 32.8?kμ was shown to account for 17.2?% of the total protein in SDS-PAGE.The immuno-reactivity of the protein was confirmed by Western-blotting using antisera against PCV2.The results suggest that the recombinant capsid protein may be used for studies on molecular diagnosis,subunit vaccine and molecular biology for PCV2.更多还原