以抗Site 1/Site 2單源抗體結合SUMO fusion蛋白所表現的口蹄疫類病毒空殼蛋白建構阻斷型ELISA

Abstract

口蹄疫病毒(Foot-and-mouth disease virus, FMDV)為無封套病毒，以正二十面體的外殼蛋白包覆一條正向單股RNA作為遺傳物質。由於其廣泛的宿主種別和具有高度的傳染性，使口蹄疫的防檢疫倍受各國政府重視並制定諸多措施防範。台灣曾於1997年爆發嚴重的口蹄疫情，僅該年度就造成約3.78億美元的損失。時至今日台灣仍未正式恢復口蹄疫非疫區的地位，加上鄰國潛在的傳播威脅，擁有一套可供快速、大量處理血清抗體的診斷平台對於防檢疫無疑都具極大助益。本研究利用knock-out mutagenesis技術與類病毒空殼蛋白(VLPs)從實驗室既有的FMDV融合瘤中篩檢能夠識別O型口蹄疫病毒中和抗體決定位的單源抗體(MAb)，考量不須操作活病毒與能兼顧抗原真實性的前提，最終使用哺乳動物細胞表現VLPs作為抗原，結合過去所篩選出的抗Site 1與Site 2 MAb，建構blocking ELSIA，復以此系統對經家畜衛生試驗所測定中和抗體力價(Serum neutralizing titer, SN titer)的97隻豬血清進行測試，結果顯示此系統與SN titer展現極高的關聯性，以抗Site 1 MAb作為tracer其相關性達到R2=0.67，在抗Site 2 MAb則為R2=0.63。根據上述結果，擬進一步拓展其應用性，結合以E.coli所生產SUMO fusion蛋白的VLPs作為抗原，藉以提升產量及降低成本，並透過大量血清樣品的測試，及生物統計分析對發揮更優化的效果，期許有朝一日能將此blocking ELISA實際應用於口蹄疫防檢疫的血清檢測。

Foot-and-mouth disease virus (FMDV) is a non-enveloped virus with an icosahedral capsid containing a positive-sense single-strand RNA. Due to its wide host range and highly contagious activity, FMD is one of the most important diseases in animal husbandry industry in the world. In 1997, a devastating outbreak of FMD in Taiwan caused losses of about US$ 378 million. Until now, FMD has not been fully eradicated in Taiwan. Hence, the establishment of a fast and stable platform for massive serologic test as a tool of prevention and control measure is undoubtedly necessary. Recently, site 1 and site 2 have been regarded as the most immune-dominant neutralization sites. In order to use monoclonal antibodies (MAbs) specifically against the well-known neutralization sites from a previously produced MAbs panel, we established a screening system based on mutated Virus-like-particles (VLPs), which alleviates biosecurity concerns that viruses pose. Due to authentic antigenicity, the mammalian cell expression system, HTK, was chosen as the antigen for the MAb-based antibody blocking ELISA (bELISA). Preliminary results showed that the PI value of the bELISA tested on 97 swine serums exhibited high correlation to SN titer, with R2 values of 0.67 and 0.63 in site 1 and site 2, respectively. Considering cost and application, we intend to express our VLPs with SUMO fusion protein in E.coli, the more productive expression system. With further optimization, we believe that this bELISA platform we have established could not only be used for FMD researching, but also as a practical serological tool for FMD monitoring and prevention.