On the basis of genomic sequence of PCV-2 in the GenBank, two primers for amplification of PCV-2 specified fragments were designed and PCR assay for the detection of PCV-2 was established,the kit was used to detect PC...On the basis of genomic sequence of PCV-2 in the GenBank, two primers for amplification of PCV-2 specified fragments were designed and PCR assay for the detection of PCV-2 was established,the kit was used to detect PCV-2. The kit’s sensitivity, specificity and expiration date were tested. The result suggested the kit had high sensitivity and specificity. It could be stored at least for one year at -20℃.展开更多
参考GenBank发表的西尼罗病毒(west nile virus,WNV)的E蛋白基因序列,自行设计合成一对引物,利用RT-PCR扩增出了WMV E基因318bp片段,将其克隆入pMD18-T-Vector载体中,阳性克隆命名pMD-E,并进行序列分析。进一步亚克隆入表达载体pET-32a...参考GenBank发表的西尼罗病毒(west nile virus,WNV)的E蛋白基因序列,自行设计合成一对引物,利用RT-PCR扩增出了WMV E基因318bp片段,将其克隆入pMD18-T-Vector载体中,阳性克隆命名pMD-E,并进行序列分析。进一步亚克隆入表达载体pET-32a(+)。重组质粒pET32a-E转化BL21(DE3)感受态细胞中表达,表达产物经SDS-PAGE可检测到分子量约为32kD的目的蛋白带,经薄层扫描分析,目的蛋白占菌体总蛋白的33．1%。表达产物纯化后,Wester-blotting分析证明表达产物能被WNV的阳性血清所识别,为下一步建立以表达产物为包被抗原建立检测马的WNV的ELISA方法打下了基础。展开更多
文摘On the basis of genomic sequence of PCV-2 in the GenBank, two primers for amplification of PCV-2 specified fragments were designed and PCR assay for the detection of PCV-2 was established,the kit was used to detect PCV-2. The kit’s sensitivity, specificity and expiration date were tested. The result suggested the kit had high sensitivity and specificity. It could be stored at least for one year at -20℃.
文摘参考GenBank发表的西尼罗病毒(west nile virus,WNV)的E蛋白基因序列,自行设计合成一对引物,利用RT-PCR扩增出了WMV E基因318bp片段,将其克隆入pMD18-T-Vector载体中,阳性克隆命名pMD-E,并进行序列分析。进一步亚克隆入表达载体pET-32a(+)。重组质粒pET32a-E转化BL21(DE3)感受态细胞中表达,表达产物经SDS-PAGE可检测到分子量约为32kD的目的蛋白带,经薄层扫描分析,目的蛋白占菌体总蛋白的33．1%。表达产物纯化后,Wester-blotting分析证明表达产物能被WNV的阳性血清所识别,为下一步建立以表达产物为包被抗原建立检测马的WNV的ELISA方法打下了基础。
