本文選題：動(dòng)物生物反應(yīng)器 + 慢病毒��； 參考：《南京農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：近十幾年來,轉(zhuǎn)基因雞的制備在人們的不懈努力下,取得了長足的進(jìn)步。這一領(lǐng)域之所以能被人們廣為關(guān)注,不僅僅因?yàn)殡u是一種理想的模式動(dòng)物,更為重要的是轉(zhuǎn)基因雞可以改造成為高效生產(chǎn)人源藥用蛋白的生物反應(yīng)器。禽輸卵管生物反應(yīng)器比傳統(tǒng)工業(yè)反應(yīng)器和哺乳動(dòng)物細(xì)胞生物反應(yīng)器更加高效而廉價(jià)已經(jīng)成為業(yè)界的共識(shí)。這也使轉(zhuǎn)基因雞的研究成為科研領(lǐng)域的一大熱點(diǎn)和生物制藥行業(yè)發(fā)展的新方向。由于雞胚胎發(fā)育的特殊性,在轉(zhuǎn)基因雞生物反應(yīng)器的制備中必需使用有別于哺乳類動(dòng)物生物反應(yīng)器的特殊手段。目前看來,使用慢病毒載體制備轉(zhuǎn)基因雞是最可靠的實(shí)驗(yàn)方法。但是由于傳統(tǒng)慢病毒包裝方法的巨大缺陷,以及雞胚注射和后續(xù)篩選中存在的諸多問題,使得成功制備轉(zhuǎn)基因雞生物反應(yīng)器的案例仍然較少被報(bào)道。在本實(shí)驗(yàn)的第一部分中,我們使用了以聚乙烯亞胺(PEI)為轉(zhuǎn)染介質(zhì),以聚乙二醇6000(PEG6000)為離心濃縮介質(zhì)的慢病毒生產(chǎn)方法,并對(duì)濃縮后的慢病毒進(jìn)行滴度測(cè)定。此外也對(duì)可能影響慢病毒滴度的幾個(gè)關(guān)鍵因素如轉(zhuǎn)染初始細(xì)胞數(shù)、N/P、轉(zhuǎn)染質(zhì)粒DNA量、佐劑的選擇等的作用進(jìn)行了分析。實(shí)驗(yàn)結(jié)果說明在使用15cm培養(yǎng)皿生產(chǎn)慢病毒的條件下,轉(zhuǎn)染質(zhì)粒DNA總量10.5μg,N/P=20,PEI濃儲(chǔ)液pH7.0-9.0,轉(zhuǎn)染時(shí)293T細(xì)胞數(shù)0.5-2×107時(shí),可以獲得較好的慢病毒滴度。PEI作為一種新的轉(zhuǎn)染試劑,可以替代磷酸鈣和脂質(zhì)體用于高滴度慢病毒的生產(chǎn)。在第二和第三部分中,我們通過對(duì)現(xiàn)有雞胚開窗注射方法諸多細(xì)節(jié)的不斷優(yōu)化,以及后續(xù)的一系列檢測(cè)手段,最終成功獲得生殖腺嵌合型轉(zhuǎn)基因雞。實(shí)驗(yàn)結(jié)果說明,通過封口方式、封口膜種類、胚胎穿刺位置、開窗操作時(shí)間、開窗后的液封處理、防止污染的措施等方面的改進(jìn),我們將開窗注射孵化率提高到了 55%。在出雛個(gè)體中,轉(zhuǎn)基因嵌合型個(gè)體所占比例提高到了 41.8%。在對(duì)所獲得的31只嵌合體雞進(jìn)一步篩選后,我們最終獲得了 4公4母共8只生殖腺嵌合型轉(zhuǎn)基因雞。綜上所述,本實(shí)驗(yàn)研究表明,PEI作為一種新的轉(zhuǎn)染試劑,可以替代磷酸鈣和脂質(zhì)體用于高滴度慢病毒的生產(chǎn)。與二者相比,PEI具有更低的操作門檻和更高的性價(jià)比,從而具有很高的使用價(jià)值,在未來一定具有廣闊的使用前景。同時(shí)本實(shí)驗(yàn)也詳細(xì)介紹了一套完整的制備轉(zhuǎn)基因雞輸卵管生物反應(yīng)器的方法,希望對(duì)廣大科研人員有所幫助。
[Abstract]:In the past ten years, the preparation of transgenic chicken has made great progress under the unremitting efforts of people. The reason why this field can be widely concerned is not only because chicken is an ideal model animal, but also because transgenic chicken can be transformed into a bioreactor that can efficiently produce human medicinal protein. Avian fallopian tube bioreactor is more efficient and cheaper than traditional industrial reactor and mammalian cell bioreactor. This also makes the research of transgenic chicken become a hot spot in the field of scientific research and a new direction of the development of biopharmaceutical industry. Because of the particularity of chicken embryo development, it is necessary to use special methods different from mammalian bioreactor in the preparation of transgenic chicken bioreactor. At present, using lentivirus vector to prepare transgenic chicken is the most reliable experimental method. However, due to the huge defects of the traditional lentivirus packaging method and the problems in chicken embryo injection and subsequent screening, the successful preparation of transgenic chicken bioreactor is still rarely reported. In the first part of this experiment, we used polyvinyleneimine (PEI) as transfection medium and polyethylene glycol 6000 (PEG6000) as centrifugal concentration medium to produce lentivirus, and determined the titer of concentrated lentivirus. In addition, the effects of several key factors which may affect the titer of lentivirus such as the number of transfection cells N / P, the amount of plasmid DNA transfection, the selection of adjuvant were analyzed. The results showed that under the condition of producing lentivirus by using 15cm culture dish, a better lentivirus titer. PEI could be obtained as a new transfection reagent under the condition that the total amount of the transfected plasmid was 10.5 渭 g / N ~ (-1) N ~ (-1) N ~ (-1) N ~ (20) P ~ (20) P ~ (-1) and 293T cell number was 0.5-2 脳 10 ~ 7, respectively. Calcium phosphate and liposomes can be substituted for the production of high titer lentivirus. In the second and third parts, we successfully obtained the genitonal chimeric transgenic chicken by optimizing the details of the existing chicken embryo fenestration injection method and a series of subsequent detection methods. The experimental results show that through the improvement of the sealing mode, the type of sealing membrane, the location of embryo puncture, the operating time of window opening, the treatment of liquid seal after window opening, and the measures to prevent pollution, we have raised the hatching rate of window opening injection to 5555%. The proportion of transgenic chimeric individuals increased to 41.8%. After further screening of 31 chimeric chickens, we finally obtained 4 males, 4 females and 8 gonadal chimeric transgenic chickens. In conclusion, as a new transfection reagent, PEI can replace calcium phosphate and liposome in the production of high titer lentivirus. Compared with the two methods, PEI has a lower operating threshold and a higher performance-to-price ratio, so it has a high value of use, and will certainly have a broad application prospect in the future. At the same time, a complete method of preparing transgenic chicken fallopian tube bioreactor was introduced in detail.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q81

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 張淑靜;高譽(yù)珊;孫紅梅;許紅;王媛媛;劉謙;胡雨;吳瑩;和欣;龔小鋼;;慢病毒介導(dǎo)的小鼠PINK1基因RNAi載體的構(gòu)建及在NIH3T3細(xì)胞中的篩選[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2015年23期

2 況野;房有榮;劉麗;王蓉;嚴(yán)子琴;李海龍;孟凡國;盛清;歐文斌;;一種高效穩(wěn)定的磷酸鈣轉(zhuǎn)染HEK293T細(xì)胞的方法[J];浙江大學(xué)學(xué)報(bào)(農(nóng)業(yè)與生命科學(xué)版);2015年04期

3 程騰;李佳佳;賀小英;劉希宇;陳珊;李文達(dá);馬利兵;;磷酸鈣轉(zhuǎn)染方法的優(yōu)化[J];內(nèi)蒙古師范大學(xué)學(xué)報(bào)(自然科學(xué)漢文版);2014年06期

4 蔡晶晶;宋小平;嚴(yán)繼貴;王雅潔;;慢病毒載體包裝與生產(chǎn)方法[J];長江大學(xué)學(xué)報(bào)(自科版);2014年09期

5 程志堅(jiān);兀飛;朱振中;李浩鵬;王國毓;賀西京;;小鼠BMPRⅠb基因慢病毒載體構(gòu)建及轉(zhuǎn)染神經(jīng)干細(xì)胞[J];陜西醫(yī)學(xué)雜志;2012年07期

6 于福先;陳曉宇;朱志偉;潘建治;;一種高效制備慢病毒表達(dá)載體的方法[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2011年03期

，

本文編號(hào)：2113955