鹅细小病毒VP3基因真核表达载体的构建及其在Vero细胞中表达

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。

Construction of goose parvovirus VP3 gene eukaryotic expression vector and expression in Vero cell line

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 ex-pression vector pVAX1. The recombinant plasmid was used to transform competent Escherichia coli DH5a and the positive clones were identified by PCR and restriction enzyme digestion. Then the recombinant pVAX1-VP3 was transfected to Vero cell line with Lipofectamine~(TM) 2000. 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 con-firmed 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.