本文選題：熒光定量PCR + 質(zhì)�？截悢�(shù)�。� 參考：《北京生物制品研究所》2005年碩士論文

【摘要】：用于乙型肝炎疫苗生產(chǎn)的重組釀酒酵母工程菌2150-2-3(pHBS56-GAP347/33)含有游離質(zhì)粒,可在宿主細(xì)胞中獨(dú)立復(fù)制并表達(dá)乙型肝炎表面抗原(HBsAg)。目前在生產(chǎn)中,為了從基因水平監(jiān)控抗原表達(dá)量,對(duì)發(fā)酵細(xì)胞進(jìn)行了質(zhì)粒保有率的檢測(cè)。但該方法只能測(cè)定細(xì)胞中有無(wú)質(zhì)粒,不能對(duì)質(zhì)粒進(jìn)行定量。由于細(xì)胞內(nèi)質(zhì)�？截惖亩嗌僦苯佑绊懽罱KHBsAg的表達(dá)量,所以在生產(chǎn)過(guò)程中有必要對(duì)質(zhì)�？截悢�(shù)進(jìn)行實(shí)時(shí)定量測(cè)定。測(cè)定質(zhì)粒拷貝數(shù)的方法有很多,如氯化銫-溴化乙錠平衡梯度離心法、高效液相色譜法、核酸雜交方法等等,但它們都有各自的缺點(diǎn),有的準(zhǔn)確性不高,有的耗時(shí)長(zhǎng)不適于實(shí)時(shí)檢測(cè),本文主要介紹了應(yīng)用實(shí)時(shí)熒光定量PCR這一新興檢測(cè)技術(shù)對(duì)質(zhì)�？截悢�(shù)目的測(cè)定方法,通過(guò)提取重組釀酒酵母基因組和質(zhì)粒DNA,以釀酒酵母細(xì)胞基因組中單拷貝基因URA3為內(nèi)標(biāo),利用質(zhì)粒HBsAg基因片段和內(nèi)標(biāo)的相對(duì)數(shù)量的測(cè)定來(lái)確定每個(gè)細(xì)胞中的質(zhì)�？截悢�(shù)。該方法結(jié)果準(zhǔn)確,90min內(nèi)即可完成檢測(cè),通過(guò)對(duì)重組釀酒酵母800L罐發(fā)酵細(xì)胞進(jìn)行測(cè)定,最終質(zhì)�？截悢�(shù)為71.5個(gè)/細(xì)胞。為了驗(yàn)證該結(jié)果的可信程度,同時(shí)用DNA斑點(diǎn)雜交的方法進(jìn)行驗(yàn)證,實(shí)驗(yàn)結(jié)果與PCR方法測(cè)定結(jié)果基本一致,證明了實(shí)時(shí)熒光定量PCR測(cè)定的質(zhì)�？截悢�(shù)的正確性。 文章進(jìn)一步介紹了該檢測(cè)方法在實(shí)際應(yīng)用:對(duì)生產(chǎn)菌種進(jìn)行了亞克隆優(yōu)選,選擇16個(gè)亞克隆在三角瓶進(jìn)行連續(xù)培養(yǎng)同時(shí)檢測(cè)質(zhì)�？截悢�(shù)和抗原表達(dá)水平,試驗(yàn)結(jié)果顯示質(zhì)�？截悢�(shù)與抗原表達(dá)水平具有一致性,同時(shí)在不同亞克隆之間存在較大的差異,說(shuō)明拷貝數(shù)測(cè)定可作為菌種優(yōu)選的一種有效手段;對(duì)三批三角瓶模擬補(bǔ)料發(fā)酵和兩批生產(chǎn)補(bǔ)料發(fā)酵細(xì)胞在發(fā)酵第40小時(shí)、52小時(shí)、64小時(shí)測(cè)定
[Abstract]:Recombinant Saccharomyces cerevisiae 2150-2-3 pHBS56-GAP347 / 33) containing free plasmids can replicate and express HBsAg in host cells independently.In order to monitor the expression of antigens at the gene level, the retention of plasmids was detected in fermentative cells.However, this method can only determine whether there are plasmids in cells, and can not quantify plasmids.Because the number of plasmid copies in the cell directly affects the final HBsAg expression, it is necessary to quantify the plasmid copy number in real time during the production process.There are many methods for determining copy number of plasmids, such as cesium chloride and ethidium bromide equilibrium gradient centrifugation, high performance liquid chromatography, nucleic acid hybridization, etc.Some time-consuming methods are not suitable for real-time detection. In this paper, a new method of detecting plasmid copy number by real-time fluorescence quantitative PCR is introduced.By extracting the recombinant Saccharomyces cerevisiae genome and plasmid DNA, the single copy gene URA3 in the genome of Saccharomyces cerevisiae was used as the internal standard. The plasmid copy number was determined by the determination of the relative number of plasmid HBsAg gene fragment and internal standard.The results of this method could be completed within 90 minutes, and the final copy number of plasmid was 71.5 / cell by determining the fermentation cells of recombinant Saccharomyces cerevisiae in 800L pot.In order to verify the reliability of the results, the DNA dot blot method was used to verify the results. The experimental results were in good agreement with those of the PCR method, which proved the correctness of the plasmid copy number measured by real-time fluorescence quantitative PCR.The article further introduces the practical application of this method: the subclone selection of the producing strain was carried out, and 16 subclones were selected for continuous culture in the triangle flask to detect the plasmid copy number and antigen expression level simultaneously.The results showed that the copy number of plasmid was consistent with the level of antigen expression, and there were great differences among different subclones, which indicated that copy number determination could be used as an effective method for the selection of bacterial species.Three batches of triangulated flasks and two batches of feedstock fermentation cells were determined at the 40th hour and the 52nd hour and the 64th hour after fermentation.
【學(xué)位授予單位】：北京生物制品研究所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R37

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前3條

1 孫美蓮;茶兒茶素生物合成相關(guān)基因表達(dá)的實(shí)時(shí)熒光定量PCR分析[D];安徽農(nóng)業(yè)大學(xué);2010年

2 茍璐璐;條銹菌誘導(dǎo)的小麥轉(zhuǎn)錄因子TaWRKY基因的克隆及表達(dá)分析[D];四川農(nóng)業(yè)大學(xué);2011年

3 范燕;用實(shí)時(shí)熒光定量PCR技術(shù)鑒定普通蕎麥（Fagopyrum esculentum Moench）三體系列[D];貴州師范大學(xué);2008年

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本文編號(hào)：1764279