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標題: 假性狂犬病毒感染細胞中之肌動蛋白結合蛋白質
Actin-binding protein in pseudorabies virus-infected cells
作者: 邱希彥
關鍵字: pseudorabies virus;假性狂犬病毒;actin-binding protein;肌動蛋白結合蛋白質
出版社: 獸醫微生物學研究所
假性狂犬病毒是阿爾法泡疹病毒亞科的一員,其基因體為線狀雙股DNA,大小約為145 kbp,為豬的重要病源之一。本實驗室先前的研究發現,細胞的肌動蛋白位於假性狂犬病毒顆粒內,為了解肌動蛋白在假性狂犬病毒生活史中的角色,先前的研究也發現cytochalasin D (可抑制F-actin形成的藥物)會使假性狂犬病毒的病毒斑大小減小且病毒斑數目減少,間接顯示肌動蛋白參與病毒的生活史。延續以往的研究,我們發現,在感染假性狂犬病毒的細胞中,以間接免疫沉澱法可測得一個高分子量(~ 135 kDa)的肌動蛋白結合蛋白質(actin-binding protein);當間接免疫沉澱法中wash buffer的氯化鈉(400 mM)改為氯化鉀(400 mM),此高分子量的肌動蛋白結合蛋白質的條帶仍清晰可見。與未感染病毒之細胞對照組比較,得知此蛋白質為病毒感染細胞中所特有。另一方面far western blotting的結果也顯示病毒顆粒含有此蛋白質。此外,也對此蛋白質作微量N-端胺基酸定序,推導出可能相對應之核酸序列(21mer),再以此核酸序列為探針,進行Southern hybridization,並偵測到此actin-binding protein之基因可能落於假性狂犬病毒DNA以Bam HI限制酵素切割之第七或第八片段之位置。進一步探討此肌動蛋白結合蛋白質的基本生化性質為日後之工作。

Pseudorabies virus is a member of alpha-herpesviruses; its genome is a double-stranded DNA of about 145 kbp in length and it is one of the most significant viral pathogens of pigs. Our previous results demonstrated that cellular actin is an internal component of the pseudorabies virion. And the size as well as the number of plaque formation can be partially inhibited by cytochalasin D (a drug inhibiting F-actin formation), suggesting that actin is involved in lytic infection of pseudorabies virus (Wong and Chen, Virus Res. 56: 191-197, 1998). Recently, we found that an unidentified actin-binding protein (~135 kDa) was present in pseudorabies virus-infected cells but not in mock-infected cells. In immunoprecipitation analysis, we replaced NaCl (400 mM) with KCl (400 mM) in wash buffer, the ~135 kDa actin-binding protein was still present in pseudorabies virus-infected cells. On the other hand, far-western analysis also showed that virions contained this actin-binding protein.
For further understanding of this protein, we did isolation and N-terminal micro-sequencing of the actin-binding protein. Then the possible corresponding nucleotide sequences were deduced. We also synthesized two corresponding oligo-nucleotides and used them as probes in Southern blotting to detect the location of the unknown gene in viral genome. The results showed that the unknown gene may be located in the BamHI- fragment 7 or 8 of pseudorabies virus (TNL strain).
Appears in Collections:微生物暨公共衛生學研究所

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