摘要
以伪狂犬病毒Ea株BamHⅠ5′片段为模板,PCR扩增出约0.6kbORF-1基因完整编码区序列,将该基因克隆到pBluescriptⅡSK+中,构建重组载体pSKORF1。进一步将该片段插入真核表达载体pEGFP-C1的增强型绿荧光蛋白EGFP下游,构建真核表达载体pEGFPORF1,脂质体法转染BHK-21细胞,G418加压筛选至正常细胞完全死亡。在倒置荧光显微镜下观察,可见pEGFPORF1转染的细胞在细胞膜和细胞浆中发出较强的荧光,证明ORF-1基因在BHK-21细胞中获得了表达。
According to the sequence of pseudorabies virus （PRV） Ea strain, a pair of specific primers was designed and the fragment containing ORF-1 gene coding domain was amplified by polymerase chain reaction （PCR） using BarnH I 5＇ fragment of PRV Ea strain as template. The fragment was cloned into pBluescript II SK＋ vector to construct the recombinant plasmid pSKORF- 1. Then,the ORF-1 gene was cloned into eukaryotic expression vector pEGFP- C1, following the enhanced green fluorescent protein（EGFP）. The recombinant plasmid pEGFPORF- 1 was transformed into BHK- 21 cells with lipofectin and selected under G418, until the normal BHK - 21 cells die out. The expressed proteins were detected under inverted fluorescence microscope. The results show that pEGFPORF - 1 was expressed in BHK - 21 cells and ORF - 1 gene was expressed in the cytomembrane and cytoplasm of BHK - 21 cells.
出处
《河南农业科学》
CSCD
北大核心
2009年第3期99-102,共4页
Journal of Henan Agricultural Sciences
基金
河南省科技攻关项目(072102140004)
