SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus)具有四種結構蛋白質，包括spike glycoprotein (S protein)、small envelope protein (E protein)、membrane glycoprotein (M protein)及nucleocapsid protein (N protein)，這些結構蛋白質可能是SARS-CoV的抗原決定部位，因此具有引發免疫反應的功能。本研究建構出4株重組桿狀病毒 (Baculovirus)- Bac-SH、Bac-EH、Bac-MH及Bac-NH於昆蟲細胞內分別表現接有Histidine-tag之S (rSH)、E (rEH)、M (rMH)及N (rNH) proteins。Sf-9細胞中所表現之rEH、rMH及rNH分別分佈於胞器、細胞核及細胞膜，分佈位置的不同造成rNH之最佳表現量 (90 μg/106 cells)高於rEH (12.5 μg/106 cells)有7倍之多。rEH與rMH在本質上的差異也造成兩者在萃取程序上的差異。rNH可容易地以native binding buffer溶出，進行IMAC (Immobilized Metal Affinity Chromatography)純化後，100 ml之細胞培養液中可得2.75 mg之rNH，其純度為90%。然而rEH則需結合二階段程序及urea的添加才能有效地萃取出，進行IMAC純化後，200 ml之細胞培養液中可得0.66 mg之rEH，其純度為72%。相較之下，嚴重的蛋白質聚集 (aggregation)問題阻礙了rMH的偵測及純化。所純化之rEH及rNH將可用於SARS診斷試劑及疫苗之研發。

The capsid of SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) is composed of four structural proteins, including the spike glycoprotein (S protein), small envelope protein (E protein), membrane glycoprotein (M protein) and nucleocapsid protein (N protein). These 4 structural proteins might be the antigenic determinants of SARS-CoV, thus they might be capable of inducing immune-responses against SARS-CoV. In this study, 4 recombinant baculoviruses Bac-SH, Bac-EH, Bac-MH and Bac-NH were constructed to express 4 Histidine-tagged S (rSH), E (rEH), M (rMH) and N (rNH) proteins, respectively, in insect cells. rEH, rMH and rNH expressed in Sf-9 cell were localized in the organelle, nucleus and plasma membrane, respectively. The maximum specific yield of rNH (90 μg/106 cells) was 7-fold higher than that of rEH (12.5 μg/106 cells), probably due to the variation in their localization. The differences in the intrinsic properties also resulted in the difference in the extraction process of rEH and rMH. rNH could be readily solublized by native binding buffer, and 2.75 mg rNH could be purified from 100 ml infected cells with a purifity of 90% by Immobilized Metal Affinity Chromatography (IMAC). However, rEH required the combination of a two-stage process and the use of urea for efficient extraction, and 0.66 mg rEH was purified from 200 ml infected cells with a purity of 72% by IMAC. In contrast, serious protein aggregation hindered the detection and purification of rMH. Purified rEH and rNH could be used in the development of SARS diagnostic reagents and possibly in the development of SARS vaccines.

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