利用桿狀病毒表現系統生產豬繁殖與呼吸道綜合症病毒的醣蛋白複合體M/GP5以利疫苗開發之研究

豬繁殖與呼吸道綜合症病毒（PRRSV）為一正股RNA病毒，主要感染豬隻並造成下痢以及母豬繁殖障礙等症狀，導致家畜業嚴重的經濟損失。在對抗PRRSV之疫苗發展中，目前以此病毒之結構醣蛋白GP5以及膜蛋白M為主要抗原標的。本研究中我們利用桿狀病毒多效表現載體同時攜帶GP5以及M蛋白，並以雙效啟動子調控GP5與Ｍ蛋白質於昆蟲及哺乳動物細胞中的表現。首先，我們成功地構築及純化此系列之重組病毒，但在西方點墨法及免疫螢光染色分析中，以GP5及M之多株抗體無法清楚偵測到GP5和M蛋白。在確認病毒DNA中仍帶有GP5及M基因後，我們進一步構築能同時表現His-tagged GP5及Flag-tagged M蛋白之重組病毒。在西方墨點法分析下我們可以成功地辨認His-tagged GP5和Flag-tagged M蛋白的表現，而在免疫螢光染色中，發現GP5及M蛋白主要分布於昆蟲細胞表面。此外，透過去醣基化分析，我們確認GP5在昆蟲細胞表現系統中具有醣基化之修飾。最後以Ni-NTA column針對His-tagged GP5蛋白進行純化，但僅能純化到微量之GP5蛋白。總而言之，我們目前成功的以桿狀病毒表現系統表現PRRSV之GP5和M蛋白，未來希望可以利用此系列病毒作為生產對抗PRRSV之次單位疫苗。

關鍵字

豬繁殖與呼吸道綜合症；桿狀病毒表現系統

並列摘要

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive strand RNA virus which can cause porcine diarrhea and reproductive failure in sows. PRRSV can cause a tremendous economic loss in porcine industry. PRRSV glycorprotein GP5 and membrane protein M which is main antigen targets were used to develop the PRRSV vaccine. In this study, we used poly-cistronic baculovirus expression system to co-express GP5 and M gene. The GP5 and M gene were expressed by CMV and polyhedrin promoters so it could express both in insect cells and mammalian cells. First, we successfully constructed and purified recombinant baculovirus. The GP5 and M proteins could not be detected clearly in western blot analysis and immunofluorescence assay. However, the GP5 and M gene were detected by PCR. We further constructed GP5 with His-tag and M protein with Flag-tag into recombinant baculovirus. In western blot analysis we could detect His-tagged GP5 and Flag-tagged M protein expression. Using immunofluorescence assay, the GP5 and M proteins were also expressed in the surface of insect cell. In addition, the glycosylation of GP5 proteins were confirmed by de-glycosylation assay. Finally, the His-tagged GP5 proteins were purified by Ni-NTA column, but the resulted proteins after purification were low. We conclude that the PRRSV GP5 and M proteins could be expressed by baculovirus expression system. Therefore, in the future, we can use this proteins as a subunit vaccine to against PRRSV.

並列關鍵字

Porcine Reproductive and Respiratory Syndrome ； baculovirus expression system

參考文獻

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CHEN, X. & LIU, J. 2011. Generation and immunogenicity of transgenic potato expressing the GP5 protein of porcine reproductive and respiratory syndrome virus. J Virol Methods, 173, 153-8.

CHIA, M. Y., HSIAO, S. H., CHAN, H. T., DO, Y. Y., HUANG, P. L., CHANG, H. W., TSAI, Y. C., LIN, C. M., PANG, V. F. & JENG, C. R. 2010. The immunogenicity of DNA constructs co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus conjugated by GPGP linker in pigs. Vet Microbiol, 146, 189-99.

CHIA, M. Y., HSIAO, S. H., CHAN, H. T., DO, Y. Y., HUANG, P. L., CHANG, H. W., TSAI, Y. C., LIN, C. M., PANG, V. F. & JENG, C. R. 2011. Evaluation of the immunogenicity of a transgenic tobacco plant expressing the recombinant fusion protein of GP5 of porcine reproductive and respiratory syndrome virus and B subunit of Escherichia coli heat-labile enterotoxin in pigs. Vet Immunol Immunopathol, 140, 215-25.

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利用桿狀病毒表現系統生產豬繁殖與呼吸道綜合症病毒的醣蛋白複合體M/GP5以利疫苗開發之研究

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