【摘要】 目的 :获得血型糖蛋白A(GPA)cDNA片段 ,并构建酵母双杂合BD端表达质粒。方法 :利用RT -PCR方法 ,从K5 6 2细胞mRNA中扩增GPAcDNA片段 ,约 410bp。测序后将其克隆至pbridge载体上。用醋酸锂法将构建好的pbridge -GPA转染酵母菌AH10 9,观察其在选择性培养基上的表达情况。结果 :克隆到的 410bpGPAcDNA编码的氨基酸序列基本与公布序列相同。pbridge—GPA转染酵母菌后 ,在SD/Gal/—His/Ura培养基上出现 1mm大小白色菌落。结论 :获得了血型糖蛋白AcDNA克隆 ,pbridge—GPA对酵母无毒性 ,不能激活检测基因 ,可作为酵母双杂合系统中的靶基因。更多还原

【Abstract】 AIM: To obtain the glycopohorin A (GPA) cDNA and construct the target gene in yeast two-hybrid.METHODS: About 410 bp cDNA fragment was amplified from K562 cell by RT-PCR.After being sequenced, the GPA gene fragment was cloned into EcoR -Ⅰ- Pst Ⅰ site of pbridge to form BD ends in yeast two-hybrid system. The recombinant plasmid was transfered into yeast AH109, and the expression in the yeast was also examined. RESULTS: The amino acid sequence encoded by cloned cDNA was mostly the same as reported GPA, and about 1 mm white yeast clone grew in the selective medium after 3 d.CONCLUSION: pbridge-GPA has nontoxic to the yeast, which can serve as a target gene in yeast two-hybrid system.更多还原