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構築含螢火蟲發光酵素基因之重組桿狀病毒及其在斜紋夜蛾細胞株之表現量測
Construction of Recombinant Baculovirus Containing Luciferase Gene and Its Expression in Spodoptera liutra Cells
摘要
將北美螢火蟲(Photinus pyralis)發光酵素基因選殖至桿狀病毒傳送載體上，構築重組質體pLl392A。利用磷酸鈣沈澱法，將重組之病毒傳送載體質體DNA及野生型苜蓿夜蛾病毒(Autographa californica nuclear polyhedrosis)DNA共轉染於斜紋夜蛾細胞，可形成重組病毒。利用病毒斑純化法，進行重組病毒之純化與篩選。抽取重組病毒之胞外病毒DNA，利用多角體蛋白基因上下游之核酸序列作為引子，進行聚合每鍊鎖反應，鑑定所得之重組病毒(Acluc)含有欲選殖之螢光酵素基因。利用蛋白質電泳分析可知，重組病毒感染斜紋夜蛾細胞後24小時起，即有分子量約6 2kDa之蛋白質產生。利用luciferin、O2以及ATP作為受質，可偵測螢光酵素催化反應後所生成之螢光，而螢光酵素的量與催化反應所產生之螢光強度具有顯著的線性關係。利用螢光強度估量細胞裂解物內螢光酵素的量，有很高的靈敏度，於細胞被感染初期即可偵測到螢光酵素。
並列摘要
The luciferase gene from the North American firely, Photinus pyralis, was cloned to the AcMNPV transfer vector, to construct the recombinant plasmid, pL1392A. After co-transfection by calcium phosphate co-precipitation with wild type virus DNA to the Spodoptera litur cell, SL7B, the recombinant virus (Acluc) was generated. Plaque purification was used to screen and purify recombinant viruses. The extracellular virus DNA of the recombinant virus was extracted to identify the insertion of luciferase gene by polymerase chain reaction. SDS-PAGE analysis revealed that a protein with molecular weight of about 62 kDa was expressed after 24 h post-infection with the recombinant virus. After luciferin, O2, and ATP were added as substrates, the activity of luciferase could be detected by a luminometer in terms of fluorescence intensity. Because there are linear relationships between the amount of luciferase and fluorescence intensity, it is easy to use the intensity of fluorescence as an indicator to estimate the amount of luciferase expressed in S. litura cells.