假性狂犬病毒立即早期基因與病毒致病機制之相關性研究

Abstract

假性狂犬病毒(Pseudorabies virus, PRV)是屬於疱疹病毒科(Herpesviridae)、阿爾法疱疹病毒亞科(Alphaherpesvirinae)中之Varicellovirus屬，可引起小豬嚴重死亡率並造成成豬之潛伏感染。PRV基因體全長大約150 Kb，而病毒基因依照感染細胞後表現的前後順序可分為立即早期基因( immediate early, IE)、早期基因( early )及晚期基因( late )三群。其中立即早期基因( IE gene)在病毒複製時，具有活化早期基因及晚期基因的功能，因此在病毒致病機制上扮演了非常重要的角色。由於假性狂犬病毒只具有一個立即早期基因，因此本實驗室先前已構築了一個以綠色螢光蛋白(green fluorescence protein, GFP)基因取代IE基因之缺陷病毒株，經過連續5次病毒斑純化步驟挑選具綠色螢光之病毒斑後，將其感染PKIE細胞(能穩定表現IE 蛋白之細胞株，以提供IE基因缺陷病毒複製時之所需)，以進行此缺陷病毒株的增殖複製而製備成種毒液，並進一步進行病毒力價測定及病毒生長曲線分析。目前共分離出2株重組病毒株。此基因缺損重組病毒株感染正常PK-15細胞株時，病毒之生長明顯受到抑制，顯示IE基因功能之缺乏可導致病毒複製能力之喪失。另外，此IE基因缺損病毒株在PK細胞上之病毒力價亦明顯降低。

Pesudorabies virus (PRV) belongs to the genus Varicellovirus of the subfamily Alphaherpesvirinae and can cause severe disease in piglets leading to the latent infection in all surviving pigs. The genome of PRV consists of a double stranded linear DNA of approximately 150 kbp. The viral genes are classified into three kinetic groups, defined as immediate-early, early and late genes, on the basis of the sequential cascade regulation during the lytic cycle. The immediate-early gene of PRV activates early and late viral genes and plays an essential role in regulating viral gene expression. PRV contains only one immediate-early gene which encodes a protein of 180 kDa. We have constructed an IE gene deleted PRV mutant strain, which N-terminal 630 codons of IE gene was replaced with GFP gene. After five times of virus plaque-purification on PKIE cells (an IE180-expressing cell line), two recombinant PRV strains were obtained. The replication of the recombinant PRV was significantly inhibited on PK cells but not restricted on PKIE cells. PRV is a neurotropic virus, the replication deficient PRV holds the great potential to be an effective and safe gene transfer vector for various cells (especially neuron cells) and provides a model for the studies of human gene therapy.