【摘要】 根据已发表的鹅细小病毒(GPV)B株核苷酸序列,设计并合成了1对引物,PCR扩增GPV吉林分离株主要结构蛋白VP3基因,获得大小为1605bp的核苷酸片段。将该片段纯化后克隆入pMD18-T载体,转化感受态大肠杆菌JM109,双酶切鉴定,筛选阳性重组质粒并测序。经过酶切和连接反应,将VP3基因克隆入真核表达载体pVAX1,转化感受态大肠杆菌DH5α,筛选阳性克隆,提取质粒,进行了PCR和酶切鉴定。通过脂质体法将pVAX1-VP3转染Vero细胞,RT-PCR和间接免疫荧光法检测。结果显示,VP3基因克隆成功,与GPVB株核苷酸序列同源性为96.2%;PCR和酶切鉴定结果证实,成功构建了含VP3基因的GPV真核表达载体pVAX1-VP3。提取转染该质粒的Vero细胞RNA,RT-PCR扩增,在1000～2000bp可见一明显DNA条带;间接荧光抗体染色转染细胞,在细胞表面可见特异荧光。更多还原

【Abstract】 A pair of primers was designed and synthesized according to nucleotide sequence of goose parvovirus(GPV) B strain and VP3 gene was amplified from the DNA of goose parvovirus isolated in Jilin province,China.The PCR amplified VP3 gene was cloned into pMD18-T vector.VP3 gene was sequenced and cloned into the eukaryotic expression vector pVAX1.The recombinant plasmid was used to transform competent Escherichia coli DH5α and the positive clones were identified by PCR and restriction enzyme digestion.Then the recombinant pVAX1-VP3 was transfected to Vero cell line with LipofectamineTM2000.The expression of pVAX1-VP3 was detected by RT-PCR and indirect immunofluorescence test.The result showed that a 1 605 bp VP3 gene fragment was amplified from the GPV DNA and was 96.2% homology with the sequence of GPV B strain.PCR and restriction enzyme digestion confirmed that VP3 gene was inserted into the eukaryotic expression vector pVAX1.The 1 000-2 000 bp DNA fragment was amplified from the RNA extracted from the Vero cell line transfected with pVAX1-VP3 by the RT-PCR method and the VP3 specific protein on the cell was detected by indirect immunofluorescence test.更多还原