| 表达猪瘟兔化疫苗株E0蛋白的PK-15细胞系构建 |
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| 引用本文： | 亢文华,郝俊峰,潘春刚,宁宜宝.表达猪瘟兔化疫苗株E0蛋白的PK-15细胞系构建[J].中国畜牧兽医,2007,34(11):63-65. |
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| 作者姓名： | 亢文华 郝俊峰 潘春刚 宁宜宝 |
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| 作者单位： | 1.中牧股份实业有限公司,北京 100070；2.中国科学院生物物理研究所，北京 100101；3.中国兽医药品监察所，北京 100081 |
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| 摘 要： | 将克隆到pGEM T-easy载体中的E0基因双酶切回收后，连接到增强型绿色荧光蛋白（EGFP）中，利用LipofectamineTM2000将重组子转染PK-15、BHK-21、VERO 3种细胞，直接在荧光显微镜下用蓝光激发进行观察，在3种细胞中都可以观察到绿色荧光，而且在转染后的24、48、72 h 3个时间点上，观察到的荧光细胞数量逐渐增多，在72 h时荧光细胞的数量在3种细胞中都达到最多，总的来看，在PK 15中荧光细胞的数量最多。通过对细胞荧光的定位发现，重组质粒的荧光主要分布在胞浆内，而且细胞核周围的荧光较强，胞核与胞浆的界限明显，尤其是在PK-15细胞和VERO细胞中这种现象尤为明显。未携带E0目的DNA的pEGFP-N1 Vector转染3种细胞后，都可以观察到绿色荧光，但是整个细胞中是均匀出现荧光的。经酶切、PCR、单抗间接免疫荧光检测，PK-E0细胞中有大量的HCLV的E0表达，单纯的PK-15细胞中没有E0的表达。HCLV株在PK-E0细胞中从48h开始到72h的滴度比在PK-15细胞中的滴度要高。证明PK-E0细胞中E0蛋白的大量表达增加了HCLV在细胞中的复制。
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| 关 键 词： | 囊膜糖蛋白Ｅ0基因 克隆 序列测定 增强型绿色荧光蛋白 |
| 文章编号： | 1671-7236（2007）11-0063-03 |
| 修稿时间： | 2007-06-14 |
Construct the PK-15 Cells that Express E0 Protein of HCLV |
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| KANG Wen-hua,HAO Jun-feng,PAN Chun-gang,NING Yi-bao.Construct the PK-15 Cells that Express E0 Protein of HCLV[J].China Animal Husbandry & Veterinary Medicine,2007,34(11):63-65. |
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| Authors: | KANG Wen-hua HAO Jun-feng PAN Chun-gang NING Yi-bao |
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| Institution: | 1.China Animal Husbandry Industry Company,Beijing 100070,China;2.Institute of Biophysics Chinese Academy of Sciences,Beijing 100101,China;3.China Institute of Veterinary Drug Control,Beijing 100081,China |
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| Abstract: | E0 gene was taked back from the pGEM-T Easy-E0 recombinant plasmid cleaved by BamH I and Pst I,was cloned into the EGFP.High pure recombinant plasmid of pEGFP-E0 was transfected into PK-15 cell,BHK-21 cell,VERO cell by LipofectamineTM2000,examine the cell for fluorescence using a UV microscope.All could find the fluorescence in PK-15 cells,BHK-21 cells,VERO cells.In 24 h,48 h and 72 h,count of the the cell for fluorescence were increasing,count of the cell for fluorescence were most in PK-15 cells,BHK-21 cells in 72 hs,VERO cells.count of the cell for fluorescence were most in PK-15 cells in 72 h.Detection indicated expressed E0 protein mainly locate in the plasma of the PK-15 cells.After cleaved by BamH I and Pst I,PCR and indirect immuno fluorescence assay,detection indicated large number of expressed E0 protein are in the PK-15 cells.After PK-15 cells and PK-E0 cells infected by HCLV,the titer of HCLV was the highest in 72 h,then began to decrease.Detection indicated large number of expressed E0 protein caused increase of the titer of HCLV. |
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| Keywords: | glycoprotein E0 clone sequence analysis enhance green fluores-cent protein |
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