本文選題：瘧疾DNA疫苗 + 構(gòu)建�。� 參考：《安徽大學(xué)》2007年碩士論文

【摘要】： DNA疫苗具有誘導(dǎo)體液和細(xì)胞免疫的雙重效應(yīng)，將最有希望成為預(yù)防和治療艾滋病、瘧疾等疾病的候選疫苗，這一領(lǐng)域的快速發(fā)展也極大地增加了對(duì)藥用質(zhì)粒DNA的需求量。然而無(wú)論在產(chǎn)量還是純度上，目前實(shí)驗(yàn)室小量制備質(zhì)粒DNA的方法都不能滿(mǎn)足藥用質(zhì)粒DNA大規(guī)模制備的需求。獲取合適的質(zhì)粒DNA制備工藝已成為DNA疫苗領(lǐng)域快速發(fā)展的瓶頸。 本實(shí)驗(yàn)室構(gòu)建的瘧疾DNA疫苗經(jīng)小鼠及恒河猴試驗(yàn)表明具有很好的免疫原性，為申請(qǐng)臨床試驗(yàn)，特進(jìn)行了制備工藝的研究。發(fā)酵是質(zhì)粒生產(chǎn)的第一步，影響發(fā)酵時(shí)質(zhì)粒產(chǎn)量的主要因素包括菌株、重組質(zhì)粒和培養(yǎng)環(huán)境。為了提高發(fā)酵培養(yǎng)時(shí)工程菌質(zhì)粒的穩(wěn)定性，本研究首先構(gòu)建了帶卡那霉素抗性標(biāo)簽的工程菌pCD-Awte／DH5α。接著優(yōu)化了工程菌的發(fā)酵條件，分別在搖瓶和5L發(fā)酵罐水平考察基本培養(yǎng)基組成及來(lái)源、補(bǔ)料培養(yǎng)基組分和溫度轉(zhuǎn)換對(duì)工程菌生長(zhǎng)及質(zhì)粒產(chǎn)量的影響。結(jié)果表明在TB培養(yǎng)基組分中添加Mg~(2+)、微量元素復(fù)合物、核苷等養(yǎng)分，補(bǔ)料培養(yǎng)基由葡萄糖代替甘油，對(duì)數(shù)中期溫度由37℃升至42℃能提高質(zhì)粒的產(chǎn)量。在優(yōu)化的培養(yǎng)條件下質(zhì)粒產(chǎn)量能達(dá)122-130mg／L培養(yǎng)液。 本研究采用堿裂解法從發(fā)酵菌體中粗提取質(zhì)粒DNA。通過(guò)分級(jí)濃縮后，采用一套色譜純化工藝來(lái)去除粗提液中混有的宿主RNA、蛋白質(zhì)、基因組DNA、內(nèi)毒素、超螺旋質(zhì)粒DNA的變性體等雜質(zhì)，，以期獲得質(zhì)粒純品。這套工藝包括Sepharose 6FF分子篩層析、Plasmidselect親硫吸附層析和Source 30Q離子交換層析、G25分子篩層析四步。接著對(duì)純品中混有的雜質(zhì)進(jìn)行質(zhì)量分析，其中宿主RNA和超螺質(zhì)粒DNA的變性體采用瓊脂糖凝膠電泳法、蛋白質(zhì)采用ELISA法、基因組DNA采用熒光定量PCR法、內(nèi)毒素采用鱟試劑法來(lái)檢測(cè)。結(jié)果表明純品質(zhì)量符合Ferreira等推薦的藥用標(biāo)準(zhǔn)。
[Abstract]:DNA vaccine has the dual effect of inducing body fluid and cellular immunity. It will be the most promising candidate vaccine for the prevention and treatment of AIDS, malaria and other diseases. The rapid development of this field has greatly increased the demand for medicinal plasmid DNA. However, in terms of yield and purity, the present methods of preparing plasmid DNA in laboratory in small quantities can not meet the needs of large-scale preparation of medicinal plasmid DNA. Obtaining suitable plasmid DNA preparation technology has become the bottleneck of rapid development in the field of DNA vaccine. The malaria DNA vaccine constructed in our laboratory showed good immunogenicity in mice and rhesus monkeys. In order to apply for clinical trial, the preparation process was studied. Fermentation is the first step in plasmid production. The main factors affecting plasmid production include strain, recombinant plasmid and culture environment. In order to improve the stability of plasmids in fermentation culture, the engineering strain pCD-Awte / DH5 偽 with kanamycin resistance label was constructed. Then the fermentation conditions of engineering bacteria were optimized. The composition and source of basic culture medium were investigated at the level of shaking flask and 5L fermenter, and the effects of composition and temperature conversion of feed medium on the growth of engineering bacteria and the yield of plasmids were also investigated. The results showed that the addition of Mg2 +, micronutrient complex, nucleoside and other nutrients to the TB medium, glucose instead of glycerol in the supplement medium, and the increase of temperature from 37 鈩

本文編號(hào)：2014011