Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/93100
標題: Surface protein expression and characterization of norovirus GII.11 from Taiwan local strain of swine
利用台灣豬場分離出之諾羅病毒GII.11進行蛋白表現與特性分析
作者: 林宜瑾
I-Chin Lin
關鍵字: 諾羅病毒
大腸桿菌表現系統
Norovirus
P domain
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摘要: Noroviruses (NoVs) are important pathogens known to cause epidemic outbreaks of severe gastroenteritis in indifferently people of all ages. Based on phylogenetic analyses, porcine NoVs belong to three distinct clusters in genogroup II (GII), which is also the most widely detected genogroup in humans. The zoonotic transmission between swine and human has been previously postulated. Porcine NoVs, majorly belonged to genotype 11, 18 and 19 of GII, has previously been detected from fecal samples of swine in diarrhea or without symptoms. Our previous work investigating the genotypic distribution of NoVs in swine in Taiwan suggested that at least two different genotypes (GII.11 and GII.18) were found from asymptomatic swine fecal samples. To further understand the prevalence of infection of swine NoVs among swine in Taiwan, blood samples from different ages of swine from different swine farms were collected to evaluate the sero-prevalence. The protruding P domain of the norovirus capsid is known to contain determinants for antibody and receptor binding. Therefore, the sequence of p-domain of GII.11 from Taiwan local isolates was cloned into pET21b and pET44a with poly-histidine-tag and expressed in Escherichia coli. The purified polyhistidine-tagged P domain proteins of NoV GII.11 showed a major protein band of about 35 kDa (P protein monomer) in SDS-10% PAGE gel. Furthermore, the p proteins could be captured effectively by porcine gastric mucin. Lastly, the p proteins were subjected to detect antibody responses among swine by enzyme-linked immunosorbent assay (ELISA) and the results showed variation by different geographic locations and ages of swine. In conclusion, our study successfully produced p protein from NoV, which demonstrated high potential in the development of diagnostic and epidemiological tools for studying noroviruses.
諾羅病毒(NoVs)為現今引起人類非細菌性急性腸胃炎的主要病原。人類的諾羅病毒主要分布於基因群: GI和GII。除了人類以外,豬的諾羅病毒主要分布於同樣是基因群GII中的11、 18和19。先前研究也推測了人與豬之間感染諾羅病毒人畜共通的可能性。豬感染諾羅病毒時並不會出現明顯的臨床症狀。本實驗室過去研究中-台灣豬隻諾羅病毒的基因型分布,也從豬隻無腹瀉症狀的糞便樣本中發現了兩種以上的不同基因型(GII.11、GII.18) 。為了進一步了解台灣豬隻的諾羅病毒感染率,收集了來自不同年齡不同豬場的豬隻血液樣本,以評估血清陽性率。過去研究得知,諾羅病毒的P domain病毒殼蛋白具有抗體與受體結合的重要結構位置。因此,本篇研究將來自台灣諾羅病毒GII.11的P domain序列放置到具有poly-histidine-tag的載體pET21b和pET44a中,並且利用大腸桿菌表現系統來表現病毒重組蛋白。經由純化過後的帶有poly-histidine-tag的GII.11P domain重組蛋白在SDS-10% PAGE gel中顯示的大小為35 kDa。此外,GII.11的P domain重組蛋白可以與豬的胃液黏膜蛋白結合(mucin) 。最後,使用酵素結合免疫吸附分析不同地理位置、年齡的豬隻抗體反應。 本研究成功生產出諾羅病毒的P domain蛋白,為將來診斷或是流行病學調查的有效工具。
URI: http://hdl.handle.net/11455/93100
文章公開時間: 2018-01-23
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