【正文】 latform baculovirus expression system . Baculovirus expression system is more and more widely applied in eukaryotic expression system in recent years, which can carry a variety of foreign genes need to express in insect cells, including fungi, plants, bacteria, virus genes. Baculovirus is doublestranded DNA virus,the natural reservoir of which is insect has a high degree of species specificity, it not infected vertebrates, positively harmless to human and animal. The present study is more alfalfa crazing armyworm nuclear polygonal body virus (AcMNPV). OpIE1 and OpIE2, two promoters of the vector pMIB/v5 His which we used to construct the optogeic gene expression system are early promoter of a baculovirus, and former promoter is more stronger. The baculovirus is Orgyia pseudotsugata multicapsid nuckear polyhedrosis virus (OpMNPV),the natural host of which is the Douglas fir tussock spite of this, the two promoters will still function in the sf9 cells, sf21, LD652Y and other insects to the drive gene expression. Baculovirus early promoter can drive gene expression of cells in the absence of viral factors . So to some extent, the pMIB vector we used also has the advantages that baculovirus expression vector has. Such as: 1) Folding correctly of rebinant protein , and the formation of disulfide bond。 而本文就主要講述對這個系統(tǒng)的探索與測試所做的努力。如： 1）表達(dá)的重組蛋白能正確折疊 ，并形成二硫鍵； 2） 翻譯后可進行修飾加工如糖基化、磷酸化、酰胺化及信號肽切割等，使重組蛋白在結(jié)構(gòu)和功能上更接近天然蛋白； 3） 與其他基于 sf9昆蟲細(xì)胞的藍(lán)光誘導(dǎo)表達(dá)系統(tǒng)的構(gòu)建 II 真核表達(dá)系統(tǒng)相比， 桿狀病毒表達(dá)系統(tǒng)可以高效表達(dá)外源基因，表達(dá)量最高可達(dá)被感染 昆蟲細(xì)胞總蛋白量的 5 0 ％； 4）可以容納大片段外源基因。該桿狀病毒是黃杉毒蛾核多角體病毒（ OpMNPV），它的天然宿主是道格拉斯冷杉毒蛾。桿狀病毒表達(dá)系統(tǒng) (baculovirus expression system)是近年來應(yīng)用較多的真核表達(dá)系統(tǒng)，它可以在昆蟲細(xì)胞中表達(dá)多種外源基因，包括真菌，植物，細(xì)菌，病毒的基因。 Sf9細(xì)胞是由 . Smith 和 . Cherry 在 1983年從細(xì)胞株 IPLBSF 21 AE得來的一個克隆。 通過這個系統(tǒng)，我們在多種哺乳動物細(xì)胞中測試了光控轉(zhuǎn)錄作用。武漢 二〇一 五 年 六 月 華中農(nóng) 業(yè)大學(xué) 學(xué) 士學(xué)位論文 基于 sf9昆蟲 細(xì)胞的 藍(lán)光誘導(dǎo)表達(dá)系統(tǒng)的構(gòu)建 Construction of Bluray Induced Expression System in sf9 insect Cells 學(xué)生姓名 : 蔡澤蘄 學(xué)生學(xué)號 : 20xx30420xx07 學(xué)生專業(yè) : 生物學(xué)基地班 指導(dǎo)教師 : 徐富強 教 授 指導(dǎo)小組 : 吳 陽 博后 華中農(nóng)業(yè)大學(xué)生命科學(xué)技術(shù)學(xué)院 二 〇 一 五 年六 月 華中農(nóng)業(yè)大學(xué) 20xx屆本科畢業(yè)論文 目錄 摘 要 ..................................................................................................................................... I Abstract .............................................................................................................................. III 縮略詞表 ............................................................................................................................ IV 1 前言 ................................................................................................................................... 1 誘導(dǎo)型啟動子 ........................................................................................................ 1 ..................................................................................... 1 ..................................................................................... 1 光誘導(dǎo)啟動子 ............................................................................................. 2 VPEL222轉(zhuǎn)錄激活系 統(tǒng) ..................................................................................... 3 VPEL222系統(tǒng)在 293T細(xì)胞中的表現(xiàn) ............................................................... 4 pMIB/V5His載體 ................................................................................................ 5 .................................................................................................... 5 ....................................................................................................................... 6 實驗材料 ................................................................................................................ 6 材料與試劑 ................................................................................................. 6 ................................................................................. 6 ................................................................................................................ 7 sf9細(xì)胞的傳代培養(yǎng) ................................................................................... 7 分子克隆基本策略 ..................................................................................... 7 通用 PCR體系及程序 ............................................................................... 8 酶切體系和方法及膠回收試劑盒的使用 ................................................. 8 ......................................................................................... 9 轉(zhuǎn)化用 DH5α 感受態(tài)的制備 ........................................................ 9 轉(zhuǎn)化的步驟 ................................................................................................. 9 質(zhì)粒抽提試劑盒的使用 ........................................................................... 10 DNA的非柱回收簡單純化步驟 .................................................... 10 ...................................................................................................... 10 ................................................................................................