Development of recombinant baculoviruses for insect pest control and foreign protein expression

重組桿狀病毒在蟲害防治及外源蛋白表現的開發 中文摘要 桿狀病毒(baculoviruses)已被成功的發展為生物農藥，其中以加州苜蓿夜蛾核多角體病毒(Autographa californica multiple nuclear polyhedrosis viruses ; AcMNPVs)最常被應用；但由於緩慢的殺蟲效果限制了桿狀病毒在農業害蟲的防治應用。此外，以安全評估的考量，如何追蹤重組桿狀病毒之基因在環境中的穩定性與存在亦是十分重要的議題。因此，vAcPhsp70EGFP/Ppag90IT2重組加州苜蓿夜蛾核多角體病毒被構築，使其同時表現綠螢光蛋白(EGFP)和以色列蠍毒殺蟲蛋白(LqhIT2)，並分別以Phsp70及Ppag90啟動子啟動。結果顯示此重組病毒除能有效防治擬尺蠖幼蟲外，還可表現EGFP進行偵測已感染之病蟲。另一方面，由於珊瑚紅螢光蛋白(DsRed)能於接收太陽光後釋放出較長波長的光；因此在本論文研究中構築可同時表現EGFP 和DsRed的重組桿狀病毒vAcR-IR-G，以進一步探討EGFP 和DsRed二螢光蛋白在作為重組桿狀病毒(GMBV)追蹤劑的比較。結果顯示此重組病毒vAcR-IR-G在感染擬尺蠖、甜菜夜蛾和斜紋夜蛾幼蟲後，當其內含之DsRed蛋白表現，於日照下且不需任何其他輔助儀器，即可釋放紅螢光。因此，我們認為將此DsRed蛋白應用於偵測田間的重組桿狀病毒，將是一個有效的追蹤劑。自1983年起，桿狀病毒/昆蟲細胞表現系統即被廣泛的用來生產許多不同的生物醫療蛋白。故本研究以此系統來表現松杉靈芝(Ganoderma tsugae)中所分離出的免疫調節蛋白FIP-gts，結果依生物活性來評估，由Sf21昆蟲細胞所產生的rFIP-gts比由E.coli細胞所產生的rFIP-gts佳。Development of Recombinant Baculoviruses for Insect Pest Control and Foreign protein expression Abstract Baculoviruses have been successfully developed as biological control agents, especially Autographa californica multiple nucleopolyhedrosis viruses (AcMNPVs), which is used most frequently. However, the slow effectiveness limits their application in agriculture. In a safety standpoint, it is also important to track the genetic stability of recombinant baculoviruses and their presence in the environments. Thus, a polyhedrin-positive recombinant AcMNPV vAcPhsp70EGFP/Ppag90IT2 was constructed for larvae to express the EGFP and scorpion neurotoxin LqhIT2 under the control of Phsp70 and Ppag90 promoter, respectively. These results revealed that recombinant AcMNPV vAcPhsp70EGFP/Ppag90IT2 may significantly increase the insecticidal potency against Trichoplusia ni when additional EGFP was expressed as a visible marker. On the other hand, the red-protein pigment of DsRed, a red fluorescence protein, may convert the short wavelength light component of the solar radi¬ation into a longer wavelength light. Thus, the EGFP and DsRed fluorescent genes are also expressed simultaneously in insect larva to evaluate which fluorescent protein is relatively bright and is suitable to act as GMBV tracer. Results demonstrate the insect larvae, including Tri¬choplusia ni, Spodoptera exigua, and Spodoptera litura, infected by the genetic modified baculovirus (GMBV) containing DsRed gene can emit red fluorescence under sun light without any prosthetic apparatus. So, DsRed may serve as a powerful tracer to evaluate the efficiency of GMBV as an insecticide in the field. Since 1983, the baculovirus-insect cell expression system has been widely used to produce recombinant proteins for many different biomedical applications. In this study, the baculovirus/insect cell expression system is utilized to produce active FIP-gts, a fungal immunomodulatory protein from Song-Shan Lingzhi (Ganoderma tsugae). As expected, rFIP-gts produced in Sf21 cells is a better source than that produced in E. coli cells for the application of oral administration.Table of Contents English abstract ⅰ Chinese abstract ⅲ List of Tables and Figures ⅵ Research background 1 Reference 11 Figures 16 Chapter1. Enhancing insecticidal efficacy of baculovirus by early expressing an insect neurotoxin, LqhIT2, in infected Trichoplusia ni larvae Abstract 27 Introduction 28 Materials and Methods 31 Results 35 Discussion 38 References 40 Tables 44 Figures 46 Chapter2. Coral red fluorescence protein as tracer Abstract 51 Introduction 52 Materials and Methods 54 Results and Discussion 56 References 59 Figures 61 Chapter3. Functional expression of FIP-gts, a fungal immunomodulatory protein from Ganoderma tsugae, in Sf21 insect cells Abstract 63 Introduction 64 Materials and Methods 67 Results 73 Discussion 76 References 79 Table 84 Figures 85 Conclusion and Future Prospects 90 Reference 9

METHOD FOR INHIBITING CELLULAR NA^+/K^+-ATPASE ACTIVITY

一種丹酚酸B鹽、或其異構物、前體、衍生物、其醫藥上可接受的鹽類或其組合物作為Na^+/K^+-ATP抑制劑的用途,借助一種常用之丹酚酸B鹽--丹酚酸B鎂鹽,進行Na^+/K^+-ATP活性測量,證實丹酚酸B鎂鹽的結構具備可取代强心配醣體的固醇類藥物的環戊氫菲環化合物的性質。此外,腦部缺血試驗亦證實丹酚酸B鎂鹽可有效減少腦細胞被破壞的程度。上述之功效皆由於丹酚酸B鹽可抑制Na^+/K^+-ATP主動運輸的磷酸根水解,以降低細胞的Na^+/K^+交換,並使細胞內的鈣離子增加,而產生强心、利尿、保護神經細胞凋亡、治療心臟衰弱、預防及治療中風等病症的用途