【摘要】： 近年來DNA疫苗使用質(zhì)粒DNA作為目的基因載體進(jìn)入靶細(xì)胞成為研究熱點。DNA疫苗可使外源基因在靶細(xì)胞中表達(dá),誘導(dǎo)宿主產(chǎn)生體液和細(xì)胞免疫應(yīng)答。但質(zhì)粒DNA在靶細(xì)胞中表達(dá)效率比較低,所以如何大規(guī)模生產(chǎn)符合藥用標(biāo)準(zhǔn)的質(zhì)粒DNA成為關(guān)注的焦點。 本論文對工程菌培養(yǎng)基的優(yōu)化、發(fā)酵工藝各參數(shù)的影響、堿裂解的優(yōu)化、層析純化路線及質(zhì)粒DNA質(zhì)量控制進(jìn)行深入的研究。 (1)利用響應(yīng)曲面法的Plackett-Burman法和中心組合設(shè)計對培養(yǎng)基進(jìn)行了優(yōu)化,我們確定了發(fā)酵培養(yǎng)基配方。(2)通過實驗確定了發(fā)酵各參數(shù)對質(zhì)粒DNA拷貝數(shù)影響,優(yōu)化發(fā)酵工藝。(3)設(shè)計了堿裂解反應(yīng)器,使堿裂解操作變成自動、連續(xù)化的過程,適用于工業(yè)化生產(chǎn)。(4)確定質(zhì)粒DNA層析純化路線為離子交換色譜、疏水色譜、分子排阻色譜。(5)建立與本工藝相符質(zhì)量檢測平臺,對工藝各階段能實施質(zhì)量檢測和質(zhì)量控制。 本工藝路線質(zhì)粒DNA總回收率為46%。依據(jù)“DNA疫苗臨床用藥質(zhì)量指導(dǎo)原則”進(jìn)行各項檢驗,結(jié)果符合DNA疫苗臨床用藥標(biāo)準(zhǔn)。結(jié)果表明本工藝適用于工業(yè)化生產(chǎn)符合藥用標(biāo)準(zhǔn)的質(zhì)粒DNA。
[Abstract]:In recent years, plasmid DNA has been used as a target gene vector for DNA vaccine to enter target cells. DNA vaccine can induce the expression of exogenous genes in target cells and induce humoral and cellular immune responses of the host. However, the expression efficiency of plasmid DNA in target cells is relatively low, so how to produce plasmid DNA in large scale according to medical standards has become the focus of attention. In this paper, the optimization of the culture medium of engineering bacteria, the influence of fermentation parameters, the optimization of alkali cracking, The purification route of chromatography and the quality control of plasmid DNA were studied. (1) the culture medium was optimized by using the Plackett-Burman method of response surface method and the center combination design. We determined the formula of fermentation medium. (2) determined the effect of fermentation parameters on the copy number of plasmid DNA, and optimized the fermentation process. (3) designed the alkaline cracking reactor to make the alkaline lysis operation automatic and continuous. It is suitable for industrial production. (4) the purification routes of plasmid DNA chromatography are determined as ion exchange chromatography, hydrophobic chromatography and molecular exclusion chromatography. (5) the quality detection platform consistent with this process is established, and the quality detection and quality control can be carried out in all stages of the process. The total recovery rate of plasmid DNA was 46%. According to the guiding principles of Clinical Drug use of DNA Vaccine, the results were in accordance with the standard of clinical use of DNA vaccine. The results show that this process is suitable for the industrial production of plasmid DNA. which meets the medical standard.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：Q789;R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 宋振忠;徐志光;黃炯;薛英;王延;許禮義;張讀樸;魯立柱;蔡宏;呼西旦;;牛分支桿菌DNA疫苗工程菌的高密度培養(yǎng)研究[J];動物醫(yī)學(xué)進(jìn)展;2010年05期

相關(guān)碩士學(xué)位論文 前3條

1 宋振忠;牛結(jié)核病三價DNA疫苗制備工藝研究及免疫效果初步評價[D];新疆農(nóng)業(yè)大學(xué);2010年

2 韓依辰;基因治療用質(zhì)粒DNA生產(chǎn)及產(chǎn)物純化的研究[D];長春工業(yè)大學(xué);2010年

3 朱麟;攜帶IBV基因真核表達(dá)質(zhì)粒的大腸桿菌工程菌發(fā)酵工藝研究[D];四川農(nóng)業(yè)大學(xué);2008年

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本文編號：2270746