本文關(guān)鍵詞：改良醋酸鋰轉(zhuǎn)化法在白念珠菌質(zhì)粒轉(zhuǎn)化中的實(shí)驗(yàn)研究　出處：《南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版)》2017年02期 　論文類型：期刊論文

  更多相關(guān)文章： 白念珠菌 醋酸鋰轉(zhuǎn)化法 轉(zhuǎn)化率

【摘要】：目的:通過增加白念珠菌細(xì)胞壁通透性改良傳統(tǒng)醋酸鋰轉(zhuǎn)化法的質(zhì)粒轉(zhuǎn)化率。方法:重組質(zhì)粒經(jīng)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(xué)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(zhì)粒轉(zhuǎn)化率,篩選最適宜DMSO濃度及熱休克時間,構(gòu)建改良醋酸鋰轉(zhuǎn)化法。隨后,采用選擇性培養(yǎng)法和PCR驗(yàn)證比較傳統(tǒng)醋酸鋰轉(zhuǎn)化法及改良醋酸鋰轉(zhuǎn)化法白念珠菌的質(zhì)粒轉(zhuǎn)化率。結(jié)果:發(fā)現(xiàn)采用5%的DMSO化學(xué)處理和將42℃熱休克時間調(diào)整為3 h后陽性克隆子數(shù)量增多最明顯。改良醋酸鋰轉(zhuǎn)化法的的白念珠菌質(zhì)粒轉(zhuǎn)化率為1.5×105陽性克隆子/1μg質(zhì)粒DNA/108個細(xì)胞,而傳統(tǒng)醋酸鋰轉(zhuǎn)化法轉(zhuǎn)化率為0.6×105陽性克隆子/1μg質(zhì)粒DNA/108個細(xì)胞。改良醋酸鋰轉(zhuǎn)化法的轉(zhuǎn)化率明顯高于傳統(tǒng)醋酸醋酸鋰轉(zhuǎn)化法,兩種方法質(zhì)粒轉(zhuǎn)化率的統(tǒng)計(jì)學(xué)比較存在顯著性差異。結(jié)論:增加二甲基亞砜化學(xué)處理和調(diào)整熱休克時間的改良醋酸鋰轉(zhuǎn)化法可以顯著提高白念珠菌質(zhì)粒轉(zhuǎn)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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