Establishment and Characterization of the Cell Line with Stable Expression of the Pseudorabies Virus Immediate-early Protein IE180

假性狂犬病毒（pseudorabies virus; PRV）是屬於阿爾伐疱疹病毒亞科（alphaherpesvirinae）之一員。此病毒在增殖時依據基因表現時間之先後次序可分為立即早期（immediate-early; IE）、早期（earIy）及晚期（Iate）三群基因。PRV只有一個IE基因，其產物（立即早期蛋白）為分子量180 kDa的磷酸化蛋白質，簡稱為IE180，其功能為負責啟動早期及晚期基因，是病毒複製時重要的必需基因。為了進一步研究IE180的特性，我們將含有假性狂犬病毒台灣台商分離株IE基因之真核表現質體轉染到PK15細胞中，利用G418長期篩選後，共獲得4株抗藥細胞株並命名為PK/IE 1-4。PK/IE細胞經由dot blot hybridization、RT-PCR及immunoperoxidase staining等方法證實此細胞株染色體DNA含有IE基因且此基因可進而表現產生IE mRNA 及IE180蛋白質。以10^(-4) M Dexamethason處理PK/IE細胞後進行PRV病毒生長曲線分析顯示比正常PK-15細胞之病毒量約可提高3倍以上，更加證明此細胞不但能持續表現具生物活性功能的IE180蛋白質亦能促進病毒之複製而能提高病毒之產量。此一持續表現PRV IE180之細胞株將來可提供作為大量生產假性狂犬病毒之宿主細胞，並能進一步應用此種細胞株來構築IE基因缺損病毒，以對假性狂犬病毒之立即早期基因功能作更深入的研究。

關鍵字

立即早期蛋白；假性狂犬病毒；穩定表現

並列摘要

Pseudorabies virus (PRV) is an alphaherpesvirus causing severe disease in swine. The immediate-early protein (IE180) of PRV functions as a potent transactivator for viral gene transcription and plays an essential role in regulating viral gene expression. To further characterize the function and regulation of PRV IE180, a recombinant eukaryotic expression plasmid containing complete PRV IE gene under the control of the dexamethasone-inducible MMTV-LTR promoter was transfected into porcine kidney cell line (PK-15) for generating the stable IE180-expressing cells. A stable expression cell line (designated PK/IE) containing the PRV IE gene in cellular genome was obtained and characterized by dot blot hybridization. Expression of the IE180 in PK/IE cell was further confirmed by immunoperoxidase staining. Multiplication of PRV in the PK/IE cells was enhanced and the viral titer increased about threefold when the cells were treated with 10^(-4) M dexamethasone.

並列關鍵字

Immediate-early protein ； Pseudorabies virus ； Stable expression

國際替代計量

Establishment and Characterization of the Cell Line with Stable Expression of the Pseudorabies Virus Immediate-early Protein IE180

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