醫(yī)學(xué)免費(fèi)論文:pET15b�瞃ARA�睧GFP原核表達(dá)質(zhì)粒的構(gòu)建及融合蛋白YARA�睧GFP的表達(dá)與純化

增強(qiáng)型綠色熒光蛋白(Enhanced green fluorescent protein，EGFP)是綠色熒光蛋白(Green fluorescent protein，GFP)的突變體[19]。GFP的分子量為27 kD，它的表達(dá)沒(méi)有種屬特異性，不需要輔助因子、底物或其他基因表達(dá)產(chǎn)物的協(xié)助作用，靈敏度高[20]。EGFP在480 nm波長(zhǎng)的激發(fā)下可以發(fā)射出比GFP亮35倍的綠色熒光，使得其觀察和檢測(cè)更為準(zhǔn)確。本研究采用的EGFP可以直接在熒光顯微鏡下對(duì)轉(zhuǎn)導(dǎo)的活體細(xì)胞進(jìn)行觀察，觀察后的細(xì)胞可繼續(xù)用于后續(xù)的實(shí)驗(yàn)，為YARA介導(dǎo)的融合蛋白轉(zhuǎn)導(dǎo)能力的研究提供了一種理想的目的蛋白。

本研究通過(guò)基因重組的方法構(gòu)建出原核表達(dá)質(zhì)粒pET15b�瞃ARA�睧GFP，轉(zhuǎn)化大腸桿菌BL21(DE3)，然后用IPTG誘導(dǎo)表達(dá)出融合蛋白YARA�睧GFP。根據(jù)融合蛋白的N端帶有6個(gè)組氨酸標(biāo)簽，利用鎳柱金屬螯合親和層析，純化出YARA�睧GFP。純化后的蛋白可以用特異性的抗組氨酸“標(biāo)簽”抗體方便地進(jìn)行檢測(cè)。純化蛋白的SDS�睵AGE電泳和Western blot結(jié)果顯示YARA�睧GFP分子量約為29 kD，與預(yù)測(cè)的相符。本研究為觀察YARA介導(dǎo)的EGFP在離體和在體的轉(zhuǎn)導(dǎo)能力進(jìn)而為利用YARA作為攜帶具有潛在治療作用的蛋白質(zhì)的載體研究奠定了基礎(chǔ)。

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