Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/12795
標題: 假性狂犬病毒顆粒上細胞蛋白質之研究
Study on Cellular Proteins Associated with Virions of Pseudorabies Virus
作者: 宏, 陳祈
Chen, Chi-Hung
關鍵字: Pseudorabies Virus;假性狂犬病毒;Cellular Protein;Virions;細胞蛋白質;病毒顆粒
出版社: 獸醫學系
摘要: 
摘要假性狂犬病毒是α-herpesviruses的一員,為豬的重要病源之一。我
們已成功的藉由蔗糖梯度離心,純化出假性狂犬病毒顆粒。以限制酵素
Kpn I分解病毒DNA、Western blotting偵測病毒醣蛋白gE,證實完整的病
毒顆粒位於蔗糖梯度離心後之第四分層物。在蔗糖梯度離心後之第二、第
三、第四分層物,含有宿主蛋白actin。為了了解actin在假性狂犬病毒顆
粒的那一位置,我們先以trypsin分解病毒封套上的polypeptide,或以
Triton X-100去除病毒的封套後,再以Western blotting偵測,結果顯示
仍能偵測到actin,意謂actin在假性狂犬病毒顆粒內。為了了解actin在
假性狂犬病毒生活史中扮演何者角色。我們在病毒感染MDBK細胞後,加入
cytochalasin D (可抑制F-actin的形成),接著觀察假性狂犬病毒斑的形
成。結果顯示cytochalasin D將使假性狂犬病毒感染MDBK細胞後,形成的
病毒斑大小減小與病毒斑數目減少。另一方面,我們也發現熱休克蛋白70
出現在第二、第三、第四分層物。而ERK-2 (extracellular-regulated
kinase 2 )出現在第二、第三分層物。以Triton X-100移去病毒封套後,
Western blotting便偵測不到熱休克蛋白70,因此我們認為它可能位於病
毒封套的外圍。綜合以上的結果,我們認為在假性狂犬病毒生活史中,宿
主細胞的某些蛋白可能扮演重要的角色。

Pseudorabies virus (PrV), a member of alpha-herpesviruses, is
one of the most significant viral pathogens for swine. We had
successfully purified virions of PrV by sucrose centrifugation
gradient. With Kpn I restriction enzyme digestion of viral DNA
and Western blot of viral glycoprotein E , it was demonstrated
that mature PrV virions were located in fraction IV of sucrose
gradient. Initially, actin was found in fraction II, III and IV
of gradient. In order to know the location of actin, virions
were treated either with trypsin to digest the polypeptide
moiety outside the viral envelope or with detergent Triton X-100
to remove the envelope of PrV prior to Western blotting. And we
found that viral associated actin was resistant to trypsin
digestion, indicating that actin is located inside the virion.
To understand how actin plays a role in PrV life cycle, we added
cytochalasin D, a drug inhibiting F actin formation, to PrV-
infected MDBK cells, then the plaque formation of PrV was
examined. The results showed that cytochalasin D could reduce
the size and number of PrV plaques in MDBK cells. On the other
hand, we also found that heat shock proteins 70 ( HSP70 ) were
present in all three fractions and the extracellular-regulated
kinase 2 ( ERK- 2 ) could be detected in fraction II and III.
Following Triton treatment, the heat shock protein was not
detectable by Western blotting, suggesting that it was outside
to the viral envelope. Taken together, our results suggested
that certain cellular proteins may play important roles in PrV
life cycle.
URI: http://hdl.handle.net/11455/12795
Appears in Collections:獸醫學系所

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