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標題: Development of discriminating ELISA diagnostic reagents to classical swine fever virus (CSFV) and characterization of swine U6 promoter for short hairpin RNA expression
作者: 吳經緯
Ching-Wei Wu
關鍵字: 豬瘟病毒
classical swine fever virus
enzyme-linked immunosorbent assay
short hairpin RNA
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摘要: Classical swine fever virus (CSFV) belongs to the genus Pestivirus of the Flaviviridae family. CSFV infection causes a highly contagious and severe disease resulting in large economic losses in the pig industry worldwide. Systematic vaccination and non-vaccination stamping-out policy are the two main strategies to control CSF. Recently, CSF E2 subunit marker vaccines and companion enzyme-linked immunosorbent assays (ELISAs) detecting the antibody to Erns have been considered a discriminating strategy for differentiating infected from vaccinated animals (DIVA). The purpose of this study was to produce specific monoclonal antibodies (MAbs) to Erns and E2 for further developing as diagnostic ELISA kits. The MAb CW813 was shown to specifically recognize Pichia pastoris yeast-expressed Erns (yErns), and the MAb CW813 and yErns based indirect sandwich ELISA showed the sensitivity of 96.6% (28/29) and specificity of 100% (25/25) that may provide an alternative method for developing a discriminating diagnostic kit. In addition, the MAb 1B6 was specific to yeast-expressed E2 (yE2), and panels of swine sera were tested by peroxidase-conjugated MAb 1B6-based blocking ELISA using yE2 N330 subunit as coated antigen. The assay demonstrated the sensitivity of 100% (35/35) and specificity of 100% (17/17) that may provide the routine serodiagnosis to swine antibody against CSFV E2. Further comparison of the MAb-1B6-based blocking ELISA with other commercial ELISA kits using a great amounts of field swine sera showed the sensitivity of 89.1% (49/55) and specificity of 93.9% (31/33) for the IDEXX E2 blocking ELISA identified sera, and the sensitivity of 84.9% (140/165) and specificity of 95.1% (117/123) for the BioChek E2 indirect ELISA determined samples. Moreover, expression of short hairpin RNAs (shRNAs) by the RNA polymerase type III U6 promoter is an effective and widely used strategy for RNA interference (RNAi) which has become a powerful biological tool for studying gene function and fighting viral disease by targeting the mRNA of viral genes. In this study, the swine U6 promoter driven shRNA expression system was constructed, and the transcription efficiency of swine U6 was analyzed to compare with its human or mouse homologue. The swine U6 promoter is stronger than the human or mouse homologue and can efficiently mediate RNA interference in these three different species of mammalian cell lines. The study result also revealed that the shRNA expressed by the swine U6 promoter could mediate efficient RNAi in reducing CSFV replication by 90.7%. The shRNA expression vector driven by the swine U6 promoter might be widely used for vector-based RNAi-induced gene silencing, especially to inhibit replication of other important swine virus. Taken together, the monoclonal antibodies specific to CSFV glycoproteins and developed ELISA methods for discriminating serodiagnosis as well as the anti-CSFV shRNA expression driven by swine U6 promoter may provide useful diagnostic reagents and strategies for studies and controls of CSFV with easy manipulation and low coat.
豬瘟病毒(classical swine fever virus, CSFV)屬於黃病毒科(Flaviviridae)之瘟疫病毒屬(Pestivirus),豬隻感染豬瘟病毒可引發高度傳染性及嚴重疾病導致養豬業極大的經濟損失。而目前對於豬瘟病毒之防治策略包括施打豬瘟減毒疫苗或全面撲殺受病毒感染豬隻。現今亦已發展使用豬瘟病毒醣蛋白E2標識次單位疫苗(E2 subunit marker vaccine)並利用偵測病毒醣蛋白Erns抗體的酵素連結免疫吸附分析法(enzyme-linked immunosorbent assay, ELISA)而建立一個能區別診斷受野外病毒感染與疫苗接種之動物(differentiate infected from vaccinated animals, DIVA),可針對受野外病毒感染的豬隻進行清除的防治策略。本研究之目的為製備出能辨識豬瘟病毒醣蛋白Erns與E2之特異性單株抗體並將其應用於ELISA之開發。單株抗體CW813能夠辨識以酵母菌表現之Erns抗原且以此抗體所建立之間接三明治型ELISA檢測豬隻血清顯示良好的敏感性(96.6%)與特異性(100%),可發展做為抗體區別診斷套組。而單株抗體1B6可特異性辨識酵母菌表現之E2醣蛋白,且以此抗體連結過氧化酵素所建立之阻斷型ELISA檢測豬隻血清顯示其敏感性與特異性皆為100%,可做為例行豬瘟E2抗體檢測之血清學診斷方法。進一步以此E2 blocking ELISA與市售商業套組進行野外豬隻血清之測定比較,依照市售阻斷型ELISA (IDEXX)測定之結果於本方法所測得之敏感性為89.1%且特異性為93.9%;而以市售間接型ELISA (BioChek)測定之結果於本方法所測得之敏感性為84.9%且特異性為95.1%。此外,利用RNA polymerase III的U6啟動子所誘導shRNA (short hairpin RNA)之表現系統已成為有效且廣泛用於表現干擾性核醣核酸(RNA interference, RNAi)的方式,並可做為基因功能或抗病毒的研究工具。本研究另外建立了豬U6啟動子誘導shRNA之表現系統並進行特性分析與抗病毒研究。結果顯示,以豬U6啟動子誘導shRNA表現之能力優於人或小鼠U6啟動子且將其應用於抑制豬瘟病毒複製之實驗結果亦顯示良好的抑制效果。本研究不僅製備了豬瘟病毒醣蛋白Erns與E2之特異性單株抗體進而建立了ELISA抗體區別檢測方法,亦建立了豬U6啟動子誘導shRNA之表現系統,這些研究成果將可提供對於豬瘟病毒之良好研究工具與診斷試劑,可望有助於豬瘟防疫。
文章公開時間: 2017-01-29
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