本文選題：萊姆病 + 伯氏疏螺旋體　； 參考：《昆明醫(yī)學(xué)院》2011年碩士論文

【摘要】：研究目的： 1.以伯氏疏螺旋體標(biāo)準(zhǔn)株B31株基因組DNA為模板,PCR擴增bmpA全基因序列,定向克隆和高效表達重組BmpA并純化。 2.用ELISA評估rBmpA在萊姆病血清學(xué)診斷中的敏感性和特異性。 研究內(nèi)容： 1. bmpA基因的定向克隆與重組菌的產(chǎn)生：以伯氏疏螺旋體標(biāo)準(zhǔn)株B31株基因組DNA為模板,設(shè)計定向克隆引物,PCR擴增bmpA全基因序列,將bmpA基因定向克隆入表達載體pGEX-6p-1,酶切鑒定,轉(zhuǎn)化大腸桿菌BL21菌株,獲得bmpA重組菌。 2. rBmpA高效表達與純化：從重組菌培養(yǎng)溫度,誘導(dǎo)時間,誘導(dǎo)劑的劑量,OD600等方面優(yōu)化誘導(dǎo)條件,找到高效表達rBmpA的最佳方案。用GSH柱純化rBmpA,探索純化rBmpA的最佳條件。 3. rBmpA在萊姆病診斷中的初步應(yīng)用：用萊姆病螺旋體感染的人和健康自愿者的血清,通過ELISA來評估rBmpA在萊姆病螺旋體感染診斷中的敏感性和特異性。 研究結(jié)果： 1.在基因水平和蛋白水平上,都出現(xiàn)了目的條帶和目標(biāo)峰,確定表達載體bmpA- pGEX-6p-1構(gòu)建成功,并表達rBmpA。 2.通過分析比較,用LB培養(yǎng)基培養(yǎng)重組菌,在37℃培養(yǎng),當(dāng)OD值為0.5-1.0加入終濃度為0.1mmol/ml的IPTG,誘導(dǎo)6小時,GST-BmpA融合蛋白表達量達到最大。 3.在最佳表達條件下1L的重組菌能純化到2.9-3.1mg的rBmpA蛋白。 4. rBmpA作為抗原輔助診斷萊姆病的特異性和敏感性有待進一步探索。 結(jié)論： 1.在本實驗室人員的努力下,成功的構(gòu)建了表達rBmpA的大腸桿菌原核表達系統(tǒng),并且本研究在基因水平和蛋白水平上得到鑒定。 2.找到了高效表達rBmpA的最佳方案。用GSH柱純化rBmpA,探索純化rBmpA的最適條件。 3. rBmpA作為抗原輔助診斷萊姆病的特異性和敏感性有待進一步探索。
[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，

本文編號：1977076