以桿狀病毒表現系統生產輪狀病毒VP7蛋白表現之研究

Expression of Rotavirus VP7 Protein in Baculovirus Expression Vector System

沈永智(Yung-Chih Shen)

指導教授 ： 吳宗遠

中原大學/工學院/生物科技研究所/碩士(2012年)

https://doi.org/10.6840/cycu201200267

摘要

輪狀病毒是主要嬰兒及五歲以下小孩產生腹瀉的主要原因，每年因感染輪狀病毒而死亡的小孩將近有600,000萬人。過去的研究中發現，輪狀病毒之醣蛋白VP7是疫苗主要的標的抗原。而恆河猴的輪狀病毒VP7蛋白，能夠於桿狀病毒表現系統表現，但是人類的VP7蛋白在此系統中，表現量卻相當微弱，甚至不表現的情形。在我們過去的研究中證實，輪狀病毒野生型VP7-G9在桿狀病毒表現系統中無法表現，但是經由在胺基酸序列第223的位置，將原本E突變為K後，蛋白質卻能在細胞外產生大量表現的情形。我們也發現，此蛋白質分泌之路徑，主要是經由內質網與高基氏體，將蛋白質分泌至細胞外。根據這些發現，我們更進一步探討不同型態之輪狀病毒VP7，G1、G2、G3及G4，在桿狀病毒表現系統中表現之情形。我們發現，VP7-G1、G3及G4在桿狀病毒表現系統中確實能夠微量的表現，但是G2型態卻沒辦法在桿狀病毒表現系統中表現。我們更進一步利用site-directed mutagenesis 於四種型態之VP7在相同位置上進行點突變，經由點突變後之VP7蛋白G1、G3及G4仍然可以在細胞外表現，但其表現量卻比野生型態有減少趨勢，而G2型態在點突變後仍然無法在桿狀病毒表現系統中表現。我們證實，此突變點確實可以使輪狀病毒VP7蛋白結構變得更穩定，但對於輪狀病毒VP7蛋白在桿狀病毒表現系統中表現並無顯著之影響。

關鍵字

輪狀病毒 ； 桿狀病毒表現系統

並列摘要

Rotavirus is the leading cause of severe diarrhea disease in infants and young children worldwide and resulting in nearly 600,000 children dead annually. The glycoprotein VP7, the major serotype antigen of rotaviruses, is reported as a target antigen for vaccination. In the previous studies, the rhesus strain of rotavirus （RRV） protein VP7 can be well expressed in baculovirus expression vector system （BVES）, but weak or no detectable expression for human rotavirus strain. In our previous work, we demonstrated that the wild-type of human rotavirus G9 strain VP7 could not be expressed in BVES. However, we accidentally isolated a mutant G9 strain VP7 with a point mutation on 223th residue （K223E）, which could be efficiently expressed. In addition, we also found the distribution of mutant G9 VP7 both in cytoplasm and virus infected medium, which showed the capacity of secretion of mutant G9 VP7. We then demonstrated that the route of protein secretion is through ER-Golgi pathway. Based on these findings, I expressed various strains of human rotavirus VP7 in BEVS, including G1, G2, G3 and G4 strains. By immunoblot analysis, we detected the weak signals of wild-type of VP7 in G1 , G3 and G4 strains but not in G2 strains. We used site-directed mutagenesis to create the same point mutation as the mutant G9 VP7 on the VP7 gene of the other four strains.Based on western blotting analysis, we discovered that the expression of mG1, G3 and G4 type VP7 was slightly decreasing compared to wild-type VP7, and G2 type still didn’t be expressed after mutation. We confide that the position of the mutation site is important for stability of each VP7 protein structure, but that the position of the mutation site didn’t have significant to rotavirus VP7 prtoein expression.

並列關鍵字

rotavirus ； VP7 ； baculovirus expression vector system

參考文獻

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