日本血吸虫DNA疫苗的研究和应用

发布时间：2018-01-14 22:00

本文关键词：日本血吸虫DNA疫苗的研究和应用　出处：《华中科技大学》2007年硕士论文　论文类型：学位论文

【摘要】： 血吸虫病是一种世界性的传染性疾病,它危害着人类的生命健康,阻碍社会发展。血吸虫DNA疫苗是预防血吸虫疾病的一种新方法,日本血吸虫的DNA疫苗的研究取得了很大的进展。本文克隆了日本血吸虫的抗原基因;对抗原基因进行基因融合;构建日本血吸虫DNA疫苗;研究了日本血吸虫DNA疫苗的发酵工艺,分离纯化工艺;研究了日本血吸虫DNA疫苗在体内、体外表达的可行性。主要取得以下成果: 1)克隆了四种日本血吸虫抗原基因sj23、sjFABP、sjGAPDH、sj26,经过酶切、测序验证阳性克隆子; 2)采用融合PCR实验技术,构建了两个融合基因sj23.FABP和sj26.GAPDH; 3)亚克隆四种日本血吸虫抗原基因sj23、sjFABP、sjGAPDH、sj26和两种日本血吸虫抗原基因的融合基因sj23.FABP、sj26.GAPDH,插入到真核表达载体pVIVO2的多克隆位点,构建了14种日本血吸虫DNA疫苗; 4)优化了日本血吸虫DNA疫苗的发酵条件:利用单因子实验,从5种碳源、6种氮源中最优碳源为甘油,最优氮源为豆饼汁;最佳K2HPO4添加比例为20%;通过“Plackeet-Burman”实验从6种发酵培养基的成份中筛选出3种重要影响因子:豆饼汁,牛肉汁,甘油。通过“Box-Behnken”分析,获得最佳培养基配方(m/v):NaCl 1%,YE 1%,磷酸盐0.3%(其中K2PO4添加比例为20%),甘油1.68%,牛肉汁2.28%,豆饼汁4.21%,初始pH值为7.0; 5)建立了日本血吸虫DNA疫苗的制备工艺:采用凝胶过滤去除RNA和杂蛋白,采用亲和离子层析去除开环质粒DNA,采用亲和吸附去除内毒素。最终产品经过质量评价,在琼脂糖凝胶电泳图谱中不可见RNA条带,杂蛋白质质量分数占0.11%,超螺旋质粒DNA含量达93.4%,总热源含量为0.85EU/(mg plasmid)。各项指标都达到了国家食品药品监督管理局对医用级DNA疫苗的技术要求。 6)通过间接荧光免疫检测,发现14种日本血吸虫DNA疫苗都分别能够在小鼠肌肉和MCF-7真核细胞中在抗原水平,成功表达。
[Abstract]:Schistosomiasis is a worldwide infectious disease, which endangers the health of human life and hinders the development of society. Schistosomiasis DNA vaccine is a new method to prevent schistosomiasis. Great progress has been made in the research of Schistosoma japonicum DNA vaccine. The antigenic gene of Schistosoma japonicum was cloned in this paper. Gene fusion of antigen gene; Construction of Schistosoma japonicum DNA vaccine; The fermentation and purification of Schistosoma japonicum DNA vaccine were studied. The feasibility of expression of Schistosoma japonicum DNA vaccine in vivo and in vitro was studied. 1) four kinds of Schistosoma japonicum antigen genes sj23, sjFABPN, sjGAPDH, sj26 were cloned, and the positive clones were confirmed by restriction endonuclease digestion and sequencing. 2) two fusion genes, sj23.FABP and sj26.GAPDH, were constructed by using fusion PCR technique. 3) subcloning of four Schistosoma japonicum antigen genes sj23, sjFABP, sjGAPDH, sj26, and two fusion genes, sj23.FABP, of Schistosoma japonicum antigen genes. Sj26.GAPDH, inserted into the polyclonal site of eukaryotic expression vector pVIVO2, was used to construct 14 DNA vaccines of Schistosoma japonicum. 4) the fermentation conditions of Schistosoma japonicum DNA vaccine were optimized. The optimal carbon source and soybean cake juice were glycerol and soybean cake juice respectively. The optimum addition ratio of K2HPO4 is 20. Through "Plackeet-Burman" experiment, three important influencing factors were selected from 6 kinds of fermentation medium: bean cake juice and beef juice. Glycerin. By "Box-Behnken" analysis, the best medium formula was obtained. The ratio of K2PO4 to K2PO4 was 20, glycerol was 1.68, beef juice was 2.28, bean cake juice was 4.21, initial pH value was 7.0; 5) the preparation process of Schistosoma japonicum DNA vaccine was established. The RNA and miscellaneous proteins were removed by gel filtration, and the open loop plasmid DNA was removed by affinity ion chromatography. Endotoxin was removed by affinity adsorption. After quality evaluation, the RNA band was not seen in agarose gel electrophoresis, and the content of miscellaneous protein was 0.11%. The content of superhelical plasmid DNA was 93.4%. The total heat source content is 0.85 EUU / mg plasmid.All indexes meet the technical requirements of the State Food and Drug Administration for medical grade DNA vaccine. 6) by indirect fluorescence immunoassay, 14 kinds of Schistosoma japonicum DNA vaccines were successfully expressed in mouse muscle and MCF-7 eukaryotic cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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