莱姆病螺旋体优势抗原BmpA分子克

发布时间：2018-06-04 10:46

  本文选题：莱姆病 + 伯氏疏螺旋体　； 参考：《昆明医学院》2011年硕士论文

【摘要】：研究目的： 1.以伯氏疏螺旋体标准株B31株基因组DNA为模板,PCR扩增bmpA全基因序列,定向克隆和高效表达重组BmpA并纯化。 2.用ELISA评估rBmpA在莱姆病血清学诊断中的敏感性和特异性。 研究内容： 1. bmpA基因的定向克隆与重组菌的产生：以伯氏疏螺旋体标准株B31株基因组DNA为模板,设计定向克隆引物,PCR扩增bmpA全基因序列,将bmpA基因定向克隆入表达载体pGEX-6p-1,酶切鉴定,转化大肠杆菌BL21菌株,获得bmpA重组菌。 2. rBmpA高效表达与纯化：从重组菌培养温度,诱导时间,诱导剂的剂量,OD600等方面优化诱导条件,找到高效表达rBmpA的最佳方案。用GSH柱纯化rBmpA,探索纯化rBmpA的最佳条件。 3. rBmpA在莱姆病诊断中的初步应用：用莱姆病螺旋体感染的人和健康自愿者的血清,通过ELISA来评估rBmpA在莱姆病螺旋体感染诊断中的敏感性和特异性。 研究结果： 1.在基因水平和蛋白水平上,都出现了目的条带和目标峰,确定表达载体bmpA- pGEX-6p-1构建成功,并表达rBmpA。 2.通过分析比较,用LB培养基培养重组菌,在37℃培养,当OD值为0.5-1.0加入终浓度为0.1mmol/ml的IPTG,诱导6小时,GST-BmpA融合蛋白表达量达到最大。 3.在最佳表达条件下1L的重组菌能纯化到2.9-3.1mg的rBmpA蛋白。 4. rBmpA作为抗原辅助诊断莱姆病的特异性和敏感性有待进一步探索。 结论： 1.在本实验室人员的努力下,成功的构建了表达rBmpA的大肠杆菌原核表达系统,并且本研究在基因水平和蛋白水平上得到鉴定。 2.找到了高效表达rBmpA的最佳方案。用GSH柱纯化rBmpA,探索纯化rBmpA的最适条件。 3. rBmpA作为抗原辅助诊断莱姆病的特异性和敏感性有待进一步探索。
[Abstract]:Objectives of the study: 1. The genomic DNA of Borrelia burgdorferi strain B31 was used as template to amplify the whole bmpA gene sequence. The recombinant BmpA was cloned and highly expressed and purified. 2. ELISA was used to evaluate the sensitivity and specificity of rBmpA in the serological diagnosis of Lyme disease. Research content: 1. Directed cloning of bmpA gene and production of recombinant bacteria: using genomic DNA of Borrelia burgdorferi standard strain B31 as template, the whole bmpA gene sequence was amplified by directed cloning primer, and the bmpA gene was cloned into expression vector pGEX-6p-1 and identified by restriction endonuclease digestion. The recombinant strain of bmpA was obtained by transformation of Escherichia coli BL21 strain. 2. High efficiency expression and purification of rBmpA: the best way to express rBmpA was found from the aspects of culture temperature, induction time, dose of inducer and so on. GSH column was used to purify rBmpA and the optimal conditions for rBmpA purification were explored. 3. The preliminary application of rBmpA in the diagnosis of Lyme disease: the sensitivity and specificity of rBmpA in the diagnosis of Lyme disease were evaluated by ELISA using the sera of patients infected with Lyme disease and healthy volunteers. Results of the study: 1. At the level of gene and protein, there were target bands and target peaks. It was confirmed that the expression vector bmpA- pGEX-6p-1 was successfully constructed and expressed rBmpA-. 2. Through analysis and comparison, the recombinant bacteria were cultured in LB medium and cultured at 37 鈩，

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