Position: Home > Articles > Construct the PK-15 Cells that Express E0 Protein of HCLV
China Animal Husbandry & Veterinary Medicine
2007,34
(11)
64-66
表达猪瘟兔化疫苗株E0蛋白的PK-15细胞系构建
作 者：
亢文华;郝俊峰;潘春刚;宁宜宝
单 位：
中牧股份实业有限公司;中国科学院生物物理研究所;中国兽医药品监察所
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
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Lipofectami-neTM2000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。
译 名：
Construct the PK-15 Cells that Express E0 Protein of HCLV
作 者：
KANG Wen-hua1,HAO Jun-feng2,PAN Chun-gang1,NING Yi-bao3(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)
关键词：
glycoprotein E0;clone;sequence analysis;enhance green fluores-cent protein
摘 要：
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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