東方果實蠅（Bactrocera dorsalis）（雙翅目：果實蠅科）卵黃蛋白之純化、分析及其抗體製備

東方果實蠅（Bactrocera dorsalis （Hendel））體內有兩種卵黃蛋白（vitellins, Vns），定名為BdVg-1和BdVg-2，其分子量分別約為48及51 kDa，本研究主要在於將此蛋白經一系列的純化過程，自卵中萃取純化出來並用以製備抗體，以供探討卵黃原蛋白（vitellogenins, Vgs）之表現。利用硫酸銨（ammonium sulfate）鹽析法（salting out）初步分離果實蠅卵中的蛋白質，以SDS-PAGE蛋白質電泳分析得知，卵黃蛋白主要在硫酸銨溶液濃度為50～70%時被沉澱出；鹽析沉澱所得之卵黃蛋白，再以離子交換層析法（ion exchange chromatography）進一步純化顯示，卵黃蛋白主要會在0.3～0.5 M氯化鈉的濃度範圍間被析出來；再將析出液濃縮後，經凝膠過濾層析法（gel filtration chromatography）做最後的純化，即獲得純化之卵黃蛋白。以純化得之卵黃蛋白為抗原，製備抗體，所得抗體以西方墨點法（Western blotting）偵測第8日齡雌、雄成蟲血液與卵研磨液，結果顯示此抗體對東方果實蠅卵黃（原）蛋白之反應具專一性，但是此抗體對48及51 kDa卵黃（原）蛋白則具交互作用，亦即對辨識兩個卵黃原蛋白不具專一性。

關鍵字

東方果實蠅；卵黃蛋白；卵黃原蛋白；抗體

並列摘要

The Oriental fruit fly (Bactrocera dorsalis (Hendel)) has two vitellins (Vns), with estimated molecular weights of 48 and 51 kDa, respectively. Vns were sequentially purified from the eggs of B. dorsalis by salting out, ion exchange chromatography, and gel filtration chromatography. By salting out the egg homogenate, SDS-PAGE analysis revealed that the Vns were mainly precipitated in a 50~70% ammonium sulfate solution. The salted-out proteins were further purified by ion exchange chromatography, and the result showed that Vns were mainly eluted with a 0.3~0.5 M sodium chloride gradient. Finally, the elutes of ion exchange chromatography were refined by running them through gel filtration chromatography to obtain purified Vns. SDS-PAGE separated the 48- and 51-kDa purified Vns, and used them as antigens to raise polyclonal antibodies. After obtaining the antibodies, Western blotting detection of the Vns and vitellogenins (Vgs) in the hemolymph of 8-day-old male and female B. dorsalis and eggs showed that the antibodies were specific to Vns/Vgs; however, there were cross-reactivities between the 48- and 51-kDa Vns/Vgs.

並列關鍵字

Oriental fruit fly ； vitellin ； vitellogenin ； polyclonal antibody

國際替代計量

東方果實蠅（Bactrocera dorsalis）（雙翅目：果實蠅科）卵黃蛋白之純化、分析及其抗體製備

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