改良醋酸锂转化法在白念珠菌质粒转化中的实验研究

发布时间：2018-01-12 18:03

  本文关键词：改良醋酸锂转化法在白念珠菌质粒转化中的实验研究　出处：《南京医科大学学报(自然科学版)》2017年02期 　论文类型：期刊论文

  更多相关文章： 白念珠菌 醋酸锂转化法 转化率

【摘要】：目的:通过增加白念珠菌细胞壁通透性改良传统醋酸锂转化法的质粒转化率。方法:重组质粒经酶切线性化后,分别检测不同二甲基亚砜(DMSO)浓度(1%、5%和10%)化学处理和热休克时间梯度(0.5、2.0、3.0和4.0 h)白念珠菌的质粒转化率,筛选最适宜DMSO浓度及热休克时间,构建改良醋酸锂转化法。随后,采用选择性培养法和PCR验证比较传统醋酸锂转化法及改良醋酸锂转化法白念珠菌的质粒转化率。结果:发现采用5%的DMSO化学处理和将42℃热休克时间调整为3 h后阳性克隆子数量增多最明显。改良醋酸锂转化法的的白念珠菌质粒转化率为1.5×105阳性克隆子/1μg质粒DNA/108个细胞,而传统醋酸锂转化法转化率为0.6×105阳性克隆子/1μg质粒DNA/108个细胞。改良醋酸锂转化法的转化率明显高于传统醋酸醋酸锂转化法,两种方法质粒转化率的统计学比较存在显著性差异。结论:增加二甲基亚砜化学处理和调整热休克时间的改良醋酸锂转化法可以显著提高白念珠菌质粒转化率。
[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，

本文编号：1415341