BEFV G與N蛋白作為快速定量檢測標的抗原性研究

本研究以牛流行熱病毒(bovine ephemeral fever virus；BEFV)之外套膜醣蛋白G (envelope glycoprotein；G)以及核蛋白N (nucleoprotein；N)作為快速定量檢測標的並分析抗原性。牛流行熱病毒為子彈型病毒科(Rhabdoviridae)暫時熱病毒屬(Ephemerovirus)，為一單負股RNA病毒。牛流行熱病毒感染後會導致牛隻急性高燒、肌肉僵直、肺氣腫及降低泌乳量，造成嚴重的經濟損失。疾病的發生遍及非洲、中東、東亞與澳洲，地處亞熱帶區域的台灣，平均每幾年爆發一次，主要的預防方式為接種疫苗，市場上以不活化疫苗為主要劑型。目前主要以細胞培養生產病毒後不活化製造，不活化疫苗對病毒含量的需求很高，因此若能在疫苗製程裡快速檢測病毒抗原含量，作為疫苗半成品之快速檢驗方式，可有效監測製程生產病毒情形、穩定疫苗製程。本研究利用原核表現系統分別生產BEFV G及N重組蛋白抗原(BEFV88G1Ec以及BEFV88NEco)，免疫兔與小鼠後收取血清，評估作為偵測病毒抗原之多株抗體的可行性。本研究成功表現BEFV G蛋白及N蛋白，收取抗BEFV血清，並分別使用西方墨點法及斑跡墨點法分析其抗原性。結果顯示，重組蛋白BEFV88G1免疫兔隻生產之抗體可辦認BEFV88G1Eco重組蛋白抗原但無法辨認完整的病毒顆粒；使用BEFV88NEco蛋白抗原免疫小鼠生產之抗體可辨識完整的病毒顆粒。同時利用image J分析斑跡墨點法結果，以光密度測定進行線性回歸分析已可達到線性決定係數(Coefficient of determination, R2)大於0.99。本研究結果發現，使用E.coli生產的BEFV N蛋白可做為檢測完整病毒抗原之標的，對於BEFV生產條件的追蹤與監測有很高的助益，可應用於生物藥品GMP製造的研發與品管工作。

關鍵字

牛流行熱病毒；不活化疫苗； dot-blot ； G蛋白； N蛋白；快速檢測

並列摘要

This study aims to develop a rapid method for the detection of bovine ephemeral fever virus (BEFV) through the antigenicity analysis of the pro-karyotic expressed glycoprotein (G) and nucleoprotein (N). Bovine ephem-eral fever virus belongs to Ephemerovirus genus of the Rhabdoviridae family with a single-negative RNA genome. BEFV endemic in Africa, the Middle East, East Asia, and Australia, it causes the symptoms including a bi-phasic fever, salivation, ocular and nasal discharge, recumbency, muscle stiffness, lameness, and anorexia. That causes the decrease in milk production. Since the inactivated virus vaccine is the major way to prevent the disease, the mass production of virus antigen is demanded. To facilitate the production process, it is needed to titer and monitor the amounts virus antigen rapidly and effi-ciently in the manufacturing process. However, only a few studies discussed the antigen targets suitable for BEFV rapid tittering in the manufacture. Here, we demonstrated the development of prokaryotic expressed BEFV G protein (BEFV88G1Eco) and N protein (BEFV88NEco). After immunized rabbits and mice respectively, the polyclonal antiserum was employed in the West-ern-blotting and dot-blotting analysis. The results showed that the rabbit polyclonal antibody generated by BEFV88G1Eco can recognize recombi-nant protein itself but can’t identify the native virus particles in the dot-blot assay. On the other hand, the mouse polyclonal antibody immunized by the BEFV88-NEco protein can recognize both the recombinant protein itself and native virus particles. The images of dot-blotting were also analyzed by im-age J for densitometry. The coefficient of determination (R2) can reach 0.99. In conclusion, by using of BEFV88NEco recombinant protein immunized polyclonal antibody and dot-blotting analysis, we had developed a quick BEFV virus antigen quantitative method. It would be a great benefit to monitoring BEFV virus antigen amounts in the R&D or in the production of the vaccine manufacturing process.