本文關鍵詞：改良醋酸鋰轉化法在白念珠菌質粒轉化中的實驗研究　出處：《南京醫(yī)科大學學報(自然科學版)》2017年02期 　論文類型：期刊論文

  更多相關文章： 白念珠菌 醋酸鋰轉化法 轉化率

【摘要】：目的:通過增加白念珠菌細胞壁通透性改良傳統(tǒng)醋酸鋰轉化法的質粒轉化率。方法:重組質粒經酶切線性化后,分別檢測不同二甲基亞砜(DMSO)濃度(1%、5%和10%)化學處理和熱休克時間梯度(0.5、2.0、3.0和4.0 h)白念珠菌的質粒轉化率,篩選最適宜DMSO濃度及熱休克時間,構建改良醋酸鋰轉化法。隨后,采用選擇性培養(yǎng)法和PCR驗證比較傳統(tǒng)醋酸鋰轉化法及改良醋酸鋰轉化法白念珠菌的質粒轉化率。結果:發(fā)現(xiàn)采用5%的DMSO化學處理和將42℃熱休克時間調整為3 h后陽性克隆子數量增多最明顯。改良醋酸鋰轉化法的的白念珠菌質粒轉化率為1.5×105陽性克隆子/1μg質粒DNA/108個細胞,而傳統(tǒng)醋酸鋰轉化法轉化率為0.6×105陽性克隆子/1μg質粒DNA/108個細胞。改良醋酸鋰轉化法的轉化率明顯高于傳統(tǒng)醋酸醋酸鋰轉化法,兩種方法質粒轉化率的統(tǒng)計學比較存在顯著性差異。結論:增加二甲基亞砜化學處理和調整熱休克時間的改良醋酸鋰轉化法可以顯著提高白念珠菌質粒轉化率。
[Abstract]:Objective: to improve the transformation rate of plasmids by improving the permeability of Candida albicans cell wall. Methods: the recombinant plasmid was linearized by enzyme digestion. Dimethyl sulfoxide (DMSO) concentrations were measured at 5% and 10%, respectively. The chemical treatment and heat shock time gradient were 0.5% and 2.0 respectively. The plasmids transformation rate of Candida albicans at 3.0 and 4.0 h, the optimum DMSO concentration and heat shock time were screened, and the modified lithium acetate transformation method was constructed. The plasmids transformation rate of Candida albicans was compared between the traditional lithium acetate transformation method and the modified lithium acetate conversion method by using selective culture and PCR. It was found that the chemical treatment with 5% DMSO and the adjustment of heat shock time at 42 鈩，

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