腸病毒七十一型表面抗原VP1及其抗原決定位胜肽之基因構築與應用

Construction and Applications of an E. coli Protein Expression System for VP1 and Its Derived Epitope Peptides of Enterovirus 71

謝倩如(Chien-Ju Hiesh)

指導教授 ： 吳正男 ； 鄭金益

中臺科技大學/健康科學院/醫學生物科技研究所/碩士(2009年)

摘要

近年來，台灣地區每年都有腸病毒流行並以腸病毒71 型為主。腸病毒七十一型病毒( enterovirus 71,EV71)通常流行於夏季，台灣地區因位在亞熱帶，全年都可能有感染病例出現，但仍以夏季為主要流行季節。而台灣地區曾在1998 年爆發腸病毒大流行造成405 名孩童感染重症、78 名孩童死亡。因此EV71是脊髓灰質炎病毒( poliomyelitis , polio )或小兒麻痺病毒 ( Poliovirus )根除之後最重要的嗜神經組織組織的腸病道病毒。
腸病毒71 型在臨床上除手口足病(hand-foot-mouth disease)外，伴隨著中樞神經系統(central nervous system , CNS)疾病，甚至造成死亡。根據研究顯示，VP1是主要的抗原，可誘發小鼠的免疫力及保護幼鼠抵抗病毒。目前針對腸病毒71 型尚無有效的藥物或疫苗可以預防或治療，而通常出現症狀時，都已經開始影響中樞神經，因此發展疫苗進行預防的工作實則更為重要。另外目前也缺乏具高度EV71專一性的抗體偵測系統。因此本實驗的目的希望利用各式VP1融合蛋白及VP1之抗原決定位胜肽( epitope peptide)，建立良好的免疫偵測系統。利用含StrepTag-II的載體，構築StrepTag-II-VP1融合蛋白基因(NTSS-VP1)，進行蛋白質表現與純化，同時與不含StrepTag-II的VP1就產量及水溶性的性質進行比較。結果發現StrepTag-II 有助VP1的產量與水溶性。以利用NTSS載體表現比較、構築VP1之抗原決定位胜肽以大腸桿菌蛋白質表現系統、以NTSS-VP1建立ELISA微孔盤系統、VP1的融合
II
蛋白及NTSS-(EV5570)Mono-Hexa基因表現的蛋白對ICR小鼠進行抗體誘發反應測試，並以Western botting分析及ELISA測試抗體效價、使母鼠交配後以100 LD50的病毒量感染新生小鼠，並紀錄每日存活率、以非洲綠猴腎上細胞(Vero cells)進行血清中和試驗。結果顯示StrepTag-II載體可能可增加蛋白水溶性，因此提高了重組基因的水溶性蛋白產量；建立了以NTSS -VP1為抗原的ELISA偵測系統；綜合致死病毒劑量的攻毒試驗與血清中和試驗結果顯示胜肽疫苗NTSS-(E5570)Tri可能是為一個新疫苗的研究方向。

關鍵字

抗原決定位胜肽 ； 腸病毒七十一型 ； Strep Tag-II ； 脊髓灰質炎病毒 ； 小兒麻痺病毒

並列摘要

Enterovirus 71 ( EV71) is usually prevalent in the summer . Since Taiwan in the subtropical region, there may be infection in the whole year. However, it is popular in the summer . In recent years, the metropolis in Taiwan there is a pandemic of enterovirus infection and enterovirus 71 in the every year. In the year 1998, Taiwan had outbreak of intestinal virus, and caused a 405 children with severe infections and 78 children died. Therefore after eradicate poliomyelitis or Poliovirus, EV71 will be the most important addicted to Nervous tissue''s enteropathy virus.
Enterovirus 71 (EV71) is the most common etiological agent detected in cases of hand-foot-and-mouth disease (HFMD) resulting in incidences of neurological complications and fatality in recent years. VP1 protein, one of EV71 coat proteins, have been indicated to have potential to act as an antigen in both the diagnosis and subunit vaccine development against EV71 in several studies. Purified recombinant VP1 protein has also been defined as a neutralization determinant, with the ability to trigger vaccine-mediated immune responses in either Coxsackievirus B3-infected mice or swine. Further studies have also demonstrated the protective effect of a VP1 subunit vaccine, produced in either bacteria or transgenic plants, against EV71 and foot-and-mouth disease virus. To improve the productivity, stability and immobilization on microplate well, we have fuse several peptide tags with VP1, and found S-Strep TagII fusion tag may enhance the solubility, stability of VP1 and be used as a ligand to be fixed on streptavidin-coated surface. We have also tested the immunogenicity of this fusion protein and used it as a ELISA system to detect antibodies in a streptavidin-based microplate form.
IV
Constructed an E. coli protein expression system, which is based on IPTG-induced, and T7 RNA polymerase-driven expression of recombinant protein fused with His, S-Tag and StrepTagII peptide tags. Constructed ELISA systems for detection anti-VP1 antibody. Synthesized gene constructs with different copy number of fusion of two VP1 epitopes, SP55 and SP70 , to make (E5570)n epitope vaccines. Expressed and purified these constructs under the same system for VP1. Used recombinant VP1 and its derivates as antigens to immunize mice to test their immunity and protection ability againt EV71 infection.
An ELISA system was constructed in a backbone of S-StrepTag II-VP1. Further application in detecting EV71-immuned sera are under investigation. Combining data obtained from both challenge with lethal dosage of virus and micro-neutralization with serum, it has been shown one of the VP1 epitope-derived construct, NTSS-(E5570)Tri, might be a good candidate for EV71 vaccine.

並列關鍵字

enterovirus 71 ； epitopes peptide ； Strep Tag-II ； polio virus

參考文獻

1. 1998. Deaths among children during an outbreak of hand, foot, and mouth disease--Taiwan, Republic of China, April-July 1998. MMWR Morb Mortal Wkly Rep 47:629-32.

3. Barnard, D. L. 2006. Current status of anti-picornavirus therapies. Curr Pharm Des 12:1379-90.

4. Barnard, D. L., V. D. Hubbard, D. F. Smee, R. W. Sidwell, K. G. Watson, S. P. Tucker, and P. A. Reece. 2004. In vitro activity of expanded-spectrum pyridazinyl oxime ethers related to pirodavir: novel capsid-binding inhibitors with potent antipicornavirus activity. Antimicrob Agents Chemother 48:1766-72.

6. Bi, L. J., Y. F. Zhou, X. E. Zhang, J. Y. Deng, Z. P. Zhang, B. Xie, and C. G. Zhang. 2003. A MutS-based protein chip for detection of DNA mutations. Anal Chem 75:4113-9.

7. Blomberg, J., E. Lycke, K. Ahlfors, T. Johnsson, S. Wolontis, and G. von Zeipel. 1974. Letter: New enterovirus type associated with epidemic of aseptic meningitis and-or hand, foot, and mouth disease. Lancet 2:112.