pCD-Awte候选疟疾DNA疫苗制备工艺的研究

发布时间：2018-06-13 12:22

  本文选题：疟疾DNA疫苗 + 构建　； 参考：《安徽大学》2007年硕士论文

【摘要】： DNA疫苗具有诱导体液和细胞免疫的双重效应，将最有希望成为预防和治疗艾滋病、疟疾等疾病的候选疫苗，这一领域的快速发展也极大地增加了对药用质粒DNA的需求量。然而无论在产量还是纯度上，目前实验室小量制备质粒DNA的方法都不能满足药用质粒DNA大规模制备的需求。获取合适的质粒DNA制备工艺已成为DNA疫苗领域快速发展的瓶颈。 本实验室构建的疟疾DNA疫苗经小鼠及恒河猴试验表明具有很好的免疫原性，为申请临床试验，特进行了制备工艺的研究。发酵是质粒生产的第一步，影响发酵时质粒产量的主要因素包括菌株、重组质粒和培养环境。为了提高发酵培养时工程菌质粒的稳定性，本研究首先构建了带卡那霉素抗性标签的工程菌pCD-Awte／DH5α。接着优化了工程菌的发酵条件，分别在摇瓶和5L发酵罐水平考察基本培养基组成及来源、补料培养基组分和温度转换对工程菌生长及质粒产量的影响。结果表明在TB培养基组分中添加Mg~(2+)、微量元素复合物、核苷等养分，补料培养基由葡萄糖代替甘油，对数中期温度由37℃升至42℃能提高质粒的产量。在优化的培养条件下质粒产量能达122-130mg／L培养液。 本研究采用碱裂解法从发酵菌体中粗提取质粒DNA。通过分级浓缩后，采用一套色谱纯化工艺来去除粗提液中混有的宿主RNA、蛋白质、基因组DNA、内毒素、超螺旋质粒DNA的变性体等杂质，，以期获得质粒纯品。这套工艺包括Sepharose 6FF分子筛层析、Plasmidselect亲硫吸附层析和Source 30Q离子交换层析、G25分子筛层析四步。接着对纯品中混有的杂质进行质量分析，其中宿主RNA和超螺质粒DNA的变性体采用琼脂糖凝胶电泳法、蛋白质采用ELISA法、基因组DNA采用荧光定量PCR法、内毒素采用鲎试剂法来检测。结果表明纯品质量符合Ferreira等推荐的药用标准。
[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 伪 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

本文编号：2014011