選殖與研究流感病毒其野生型和突變型核蓋蛋白的寡聚合作用

A型流感病毒可以感染人類，鳥類及多種哺乳類動物。它在20世紀有四次突發變種成多型人體互相感染的病原。最近更在亞洲及歐洲持續發生H5N1禽流感，其可能致死的潛在危險，已引起重度的關切，擔心它再度造成全球性流感的大流行。A型流感病毒的基因組可分為八段，每段均被核蓋蛋白(nucleoprotein)包裹著，擁有正黏液病毒科 (Orthomyxoviridae family) 的遺傳性質。藉由核蓋蛋白的寡聚合作用，其與核糖核酸 (RNA) 的鍵結作用，再加上與病毒的核糖核酸聚合酶複合體 (vRNA-dependant RNA polymerase complex : PA, PB1, and PB2) 的鍵結，最後形成病毒的核糖核蛋白複合體 (ribonucleoprotein complex, RNP) 。在核蓋蛋白的氨基酸序列上，做點突變 : E339A或R416A，可使其蛋白質之間 (NP-NP contacts) 的寡聚合作用所需的鹽橋不再作用，因而破壞核蓋蛋白的寡聚合反應，使得原本會呈現大分子聚合物 (Oligomer) 的核蓋蛋白變成單體分子 (Monomer) ! 因此，吾人先選殖出所需的野生型核蓋蛋白，再利用大腸桿菌和pET15b質體的表現系統，成功的取得水溶性且原態 (native) 的核蓋蛋白，並將核蓋蛋白經過單點突變 (E339A或R416A) 後，根據快速蛋白質液相層析的結果發現，此兩個點突變確實都能在一定程度上破壞聚合作用所需的鹽橋，使核蓋蛋白呈現單體分子!此外，吾人也藉由OD260/OD280的比值，得知這些核蓋蛋白能與去氧核糖核酸 (DNA) 有鍵結作用且無法利用高鹽溶液和去氧核糖核酸水解酶 (DNase I) 去除之。另外，結果也發現，野生型的核蓋蛋白能保有且維持多分子聚合態，然而，兩個突變型核蓋蛋白之多分子聚合態並非動力平衡，而是隨著時間趨向形成單體分子! 藉此，吾人得以計算出一定條件下，突變型核蓋蛋白之單體分子形成的平均速率以及其多分子聚合態可能的變換模式。

關鍵字

A型流感病毒；核蓋蛋白；寡聚合作用；單點突變

並列摘要

Influenza A virus can infect humans, birds, and several kinds of mammals. In the 20th century, 4 variants of the virus have arised as human transmissible pathogens. Moreover, the outbreak of H5N1 avian influenza, was recently reported in Europe and Asia. The virus has generated serious concern for its high death rates and the epidemics all over the world. The genome of influenza A virus is separated into 8 RNP segements which are coated with nucleoproteins, a common genetic property of Orthomyxoviridae family. The oligomerization and RNA-binding characteristics of nucleoproteins indicate that they can interact with vRNA-dependant RNA polymerase complex (PA, PB1, and PB2) and vRNA to form ribonucleoprotein complex, RNP. According to previous literatures, the leading factor of oligomerization in NP-NP contacts, salt-bridge between one nucleoprotein E339 and another one R416, can be broken by single point mutation (E339A or R416A), leading to the nucleoprotein oligomers switch to the monomers. Thus, we have created the same mutations on nucleocapsid protein (NP of H1N1 and H3N2), and then obtained soluble and native nucleoproteins to perform FPLC. The FPLC patterns show that the two mutations can indeed break the salt bridge of NP-NP contacts and disrupt the original oligomerization behavior of wild-type NP to form monomeric form. In addition, the nucleoprotein will bind to the DNA during purification process, which is monitored from the OD260/OD280 ratio. However, the DNA can not be dissociated or hydrolyzed completely by using high-salt buffer and DNase I. Additionally, the FPLC patterns show that the oligomerization state of the wild-type NP is retained and maintained. However, the two mutant NPs (NP-E339A and NP-R416A) are not, indicating that the mutant NP polymers will turn into NP monomers over time. Finally, we calculated the average rate of the monomer formation and characterized their possible conversion mode.

並列關鍵字

Influenza A virus ； Nucleoprotein or Nucleocapsid protein ； Oligomerization ； Single point mutation

參考文獻

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選殖與研究流感病毒其野生型和突變型核蓋蛋白的寡聚合作用

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