本文關(guān)鍵詞：賈第蟲病毒轉(zhuǎn)染載體的構(gòu)建及綠色熒光蛋白在賈第蟲體內(nèi)的表達(dá)　出處：《吉林大學(xué)》2005年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 賈第蟲 賈第蟲病毒 GFP

【摘要】：本研究以中國(guó)人源賈第蟲病毒(GLV)基因組為基礎(chǔ),構(gòu)建GLV與GFP的嵌合體,優(yōu)化GLV表達(dá)外源基因的順式作用元件;克隆賈第蟲GDH啟動(dòng)子并對(duì)其轉(zhuǎn)錄活性進(jìn)行了鑒定;構(gòu)建NEO抗性盒pGDH5/NEO/GDH3并轉(zhuǎn)染賈第蟲,通過G418篩選獲得抗性蟲株;以G418為篩選標(biāo)記,構(gòu)建賈第蟲DNA穩(wěn)定轉(zhuǎn)染載體,建立DNA穩(wěn)定轉(zhuǎn)染方法;首次將GLV高效表達(dá)外源基因的順式作用元件置于GDH啟動(dòng)子下游,構(gòu)建GLV轉(zhuǎn)錄盒,將GLV轉(zhuǎn)錄盒與NEO抗性盒串聯(lián)獲得GLV載體pGLV,線性化pGLV后電穿孔轉(zhuǎn)染賈第蟲北京株,通過G418篩選建立了抗性蟲株,經(jīng)核酸檢測(cè)發(fā)現(xiàn)線性質(zhì)粒pGLV已整合入賈第蟲基因組,而GLV和GFP的嵌合體則以雙鏈RNA的形式存在;遺傳穩(wěn)定性分析表明該抗性蟲株具有較好的遺傳穩(wěn)定性;并且證實(shí)GFP在賈第蟲體內(nèi)得到了表達(dá),并且主要以分泌蛋白的形式表達(dá),表達(dá)GFP能與兔抗GFP血清發(fā)生反應(yīng);經(jīng)(NH_4)_2SO_4沉淀、凝膠過濾層析柱純化后其純度達(dá)85.3%,蛋白質(zhì)濃度測(cè)定表明在培養(yǎng)上清中目的蛋白含量為0.56g/L。該表達(dá)系統(tǒng)具有表達(dá)量高、表達(dá)產(chǎn)物易于純化的特點(diǎn),從而為外源基因在真核細(xì)胞中的表達(dá)提供了新的方法。
[Abstract]:In this study, Chinese Giardia virus (GLV) genome based chimeras GLV and GFP, cis acting element optimization of GLV expression of exogenous gene; cloning of Giardia GDH promoter and its transcriptional activity were identified; construction of NEO resistance box pGDH5/NEO/GDH3 transfection and Giardia, selected by G418 resistant strains; using G418 as selection marker, construction of Giardia DNA stable transfection vector, establishment of DNA stable transfection method; first GLV exogenous gene expression of the cis elements in the GDH promoter to construct GLV transcription box, GLV box and NEO box series resistance transcription vector GLV was linearized pGLV pGLV after electroporation of Giardia isolates from Beijing, selected by G418 established resistant strains, the nucleic acid detection of linear plasmid pGLV was integrated into the Giardia genome, and chimeras of GLV and GFP in double stranded RNA form;. The analysis shows that the resistant strain had good genetic stability and transmission stability; confirmed that GFP was expressed in Giardia in vivo, and expressed mainly in the form of secreted protein, the expression of GFP and Rabbit anti GFP serum reacted with _2SO_4; (NH_4) precipitation, gel filtration chromatography the purity of the purified protein reached 85.3%. The concentration measurements show that the 0.56g/L. in the protein content in the culture supernatant of expression system with high expression amount, expression characteristics of easy purification of products, and a new method for exogenous gene expression in eukaryotic cells.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R346;S852.7

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 韓乾忠;柔嫩艾美耳球蟲病毒的鑒定及細(xì)胞內(nèi)分布[D];吉林大學(xué);2011年

2 張昕鑫;陰道毛滴蟲轉(zhuǎn)染載體的構(gòu)建及EGFP在陰道毛滴蟲內(nèi)的穩(wěn)定表達(dá)[D];吉林大學(xué);2008年

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本文編號(hào)：1423340