第一型及第三型登革病毒單株抗體之製備和功能探討

引發出血性登革熱的登革病毒，持續地在亞洲和南美洲的公共衛生上構成嚴重的問題。在此研究中，我們利用登革第一型和第三型的病毒對BALB/c老鼠進行免疫，並且以細胞融合技術產生對抗第一型和第三型登革病毒的單株抗體。這些單株抗體在酵素連結免疫吸附分析法以及免疫墨點法測試中對登革病毒有專一性的反應。一些單株抗體對抗病毒的非結構性蛋白，另一些對抗病毒的套膜蛋白。西方墨點分析法顯示DA6-2, DA9-5, DA10-2, DA11-13只對抗第一型的登革套膜病毒而不會與其他血清型的登革病毒交叉反應。DC36-3同樣的只對抗第三型登革套膜病毒而不會與其他血清型的登革病毒交叉反應。DC7-33和DC14-33對四型登革病毒的套膜蛋白都有所反應。DC12-33除了第四型登革病毒以外對所有血清型的登革套膜病毒皆有所反應。其中有三株單株抗體在PRNT和免疫螢光染色的測試中被證實對登革病毒的感染途徑發揮中和性效用。我們也已經利用phage display的技術確認出這些單株抗體的中和性抗原決定位。我們相信這些登革的單株抗體與其抗原決定位能夠爲偵檢試劑和疫苗的研發提供一些有用的資訊。

關鍵字

細胞融合；登革；中和性；套膜蛋白；單株抗體

並列摘要

Dengue virus (DEN), causing dengue hemorrhagic fever (DHF), still present a public health problem in Asia and Southern America. In this study, monoclonal antibodies (mAbs) against DEN-1 and DEN-3 were generated by fusing P3-NS1/-Ag4-1 mouse myeloma cells with lymphocytes from BALB/c mice immunized with purified DEN-1 and DEN-3. MAbs were identified to react specifically to the DENs by ELISA and immunoblotting analysis. Some mAbs reacted to nonstructured protein 1 (NS1) and the others reacted to envelope proteins (E proteins). Immunoblotting analysis showed that DA6-2, DA9-5, DA10-2, DA11-13 reacted only to envelope proteins of DEN-1 and did not cross-react with DEN-2, -3, or -4. DC36-3 reacted only to envelope proteins of DEN-3. DC7-33 and DC14-33 reacted to envelope proteins of all dengue serotypes. DC12-33 reacted to envelope proteins of all dengue serotypes except DEN-4. Three mAbs were further demonstrated to neutralize DEN infection by plaque reduction neutralization test (PRNT) assay. The neutralizing epitopes of these mAbs were further identified from a random peptide library displayed on phage. Immunopositive phage clones reacted specifically with these mAbs and did not react with normal mouse serum. We believe that these mAbs and epitopes of DENs will provide information for development of virus-specific serologic diagnostic reagents and vaccines.

並列關鍵字

PRNT ； hybridoma ； neutralize ； envelope protein ； monoclonal antibodies ； dengue ； phage display

參考文獻

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第一型及第三型登革病毒單株抗體之製備和功能探討

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