酵母双杂交相关方法的改良及应用  

The improvement of some methods involved in yeast two-hybrid experiments

作　　者：徐文琳[1] 廖志勇[2] 王春丽[2] 余利红[2] 王新兴[1] 尹昭云[1] 张成岗[2] 钱令嘉[1] 

机构地区：[1]军事医学科学院卫生学与环境医学研究所,天津300050 [2]军事医学科学院放射医学研究所,北京100850

出　　处：《生物技术通讯》2003年第5期372-374,共3页Letters in Biotechnology

基　　金：军事医学科学院科技创新研究启动基金(0102001);国家自然科学基金重大研究计划(90208017);国家自然科学基金(30100049)

摘　　要：对酵母双杂交实验过程中较为耗时的阳性克隆鉴定过程进行改进,以期建立一种快速有效的鉴定方法。分别采用液氮冻融法、超声破碎法、渗透压破壁法以及煮沸裂解法裂解酵母细胞,获得质粒作为PCR模板,直接测序鉴定筛选到的相互作用蛋白。以液氮冻融法和超声破碎法裂解细胞获得的质粒为模板进行PCR,得到特异的产物,测序鉴定结果明确,与经典的鉴定方法相比效果相当,但更加经济快捷;而渗透压破壁法和煮沸裂解法则效果不好。说明前两种方法可代替常规方法用于阳性克隆的鉴定,从而加快酵母双杂交实验中大量阳性克隆的筛查工作。To improve current methods involved in yeast two-hybrid experiments and establish a fast and efficient method for positive clone screening.Four cell-lysing methods,i.e.liquid nitrogen method,ultrasonic method,osmotic pressure method and boiling method were chose to lysis the yeast cell,the plasmid was extracted and used as PCR template,the acquired interaction protein was identified directly by sequencing the PCR product.Specific and effi-cient PCR was acquired when using the plasmids extracted with liquid nitrogen method and ultrasonic method,the result was similar to that with classical method,but the time and cost were both reduced.While the results of os-motic pressure method and boiling method were not satisfying.The liquid nitrogen method and ultrasonic method were suitable for extracting plasmid,followed by PCR and direct DNA sequencing,and were good substitutes for the traditional method for identifying positive clones.By applying these methods,the screening of positive clones from yeast two-hybrid experiments was accelerated.

关 键 词：酵母双杂交 阳性克隆 筛选 PCR 

分 类 号：Q782[生物学—分子生物学]