整合素β3亚基不同片段在酵母双杂交系统中的表达及对报告基因激活作用的检测  

Expression of Different Fragments of Integrin Beta 3 Subunit in the Yeast Two-hybrid System and Their Activation of Reporter Gene

作　　者：杨建波[1] 姚佳[1] 杨洁[1] 

机构地区：[1]南京大学生命科学学院医药生物技术国家重点实验室,南京210093

出　　处：《中国药科大学学报》2005年第6期584-589,共6页Journal of China Pharmaceutical University

基　　金：国家自然科学基金资助项目(No.30171094No.30271497)~~

摘　　要：目的:为建立用于筛选人血小板整合素蛋白αⅡbβ3拮抗剂的高通量药物筛选模型,对其β3亚基不同片段在酵母双杂交系统中的表达及对报告基因的激活作用进行初步研究。方法:通过PCR扩增得到6个不同长度的整合素β3亚基基因片段,包括片段A1(55E-199Q)、A2(235K-385S)、A3(4Ⅰ-454T)、A4(4Ⅰ-77S)、A5(4Ⅰ-147S)和A6(4Ⅰ-284S),分别克隆到酵母双杂交诱饵载体pAS2-1中,然后将重组质粒导入酵母双杂交菌株Y190中,检测其表达产物在酵母细胞中对报告基因的激活作用。结果:6个片段对酵母均无毒性作用,片段A1、A2、A3、A5、A6不能激活报告基因HIS3和LacZ的表达,而片段A4和一个A3的缺失突变体可以激活报告基因的表达,β3亚基N末端存在一个转录激活区。结论:在建立模型时可以使用A1、A2、A3片段,但转录激活是否是β3亚基的生理功能仍有待进一步研究确证。AIM： To construct the bait vectors containing different amino terminal fragments of integrin beta 3 subunit in the yeast two-hybrid system, and to test whether their expressed product could affect the growth of yeast cells and activate the transcription of reporter genes. METHODS： The cDNA fragments encoding different sizes of integrin beta 3 amino terminal region were amplified by PCR, then cloned into pAS2-1. These fragments included A1 （55E- 199Q）,A2（235K-385S）, A3（4Ⅰ-454T）, A4（4Ⅰ-77S）, A5 （4Ⅰ-147S） and A6（4Ⅰ-284S）. After verified by sequencing, these bait vectors were transferred into the yeast two-hybrid host Y190 to assay whether the transcription of the reporter genes was activated. RESULTS： The bait vectors of different sizes of integrin beta 3 amino terminal region were constructed successfully. It was found that all fragments were not toxic to Y190. But fragment 4Ⅰ-77S and another mutant fragment （4Ⅰ-F56＋Q） activated the expression of the reporter genes. CONCLUSION： Fragments A1, A2 and A3 could be used as the bait in the yeast two-hybrid system. The amino terminal fragment （4Ⅰ-77S） can activate the transcription of the Gal4-dependent reporter genes when it is fused to the Gal4 DNA binding domain.

关 键 词：整合素αⅡbβ3 酵母双杂交 转录激活 

分 类 号：R346[医药卫生—基础医学]