论文部分内容阅读
目的 筛选与食管癌相关基因 1(ECRG 1)编码蛋白相互作用的蛋白 ,为其功能研究奠定基础。方法 将编码ECRG 1羧基端 378个氨基酸的DNA序列插入到pGBKT7 DNA BD载体 ,与编码Gal4DNA结合结构域的DNA序列拼接做融合基因 ,将此重组质粒与已克隆到pACT2载体上的人肝脏cDNA文库 (与编码Gal4激活结构域的DNA序列融合 )共转化酵母细胞AH10 9。ECRG 1与相应的人肝脏cDNA片段编码的蛋白发生相互作用后 ,可激活报告基因的表达。排除假阳性后 ,阳性克隆中的文库cDNA进行测序分析。同源性检索搜寻GenBank中与之相同或相似的序列。结果 共转化得到约 3× 10 6个转化子 ,2 3个克隆有报告基因的表达。排除假阳性后 ,得到 2个阳性克隆 ,它们分别编码Miz 1(myc interactingznfingerprotein 1)和FLNA(actin bindingprotein 2 80 )。结论 ECRG 1基因编码蛋白在酵母中可特异性的结合Miz 1和FLNA ,提示ECRG 1可能通过与MIZ 1、FLNA相互作用参与调控细胞周期的运行。
Objective To screen the proteins that interact with the protein encoded by esophageal cancer-associated gene 1 (ECRG 1), and lay the foundation for its function. Methods The 378 amino acid sequence encoding the carboxyl terminal of ECRG 1 was inserted into pGBKT7 DNA BD vector and fused with the DNA sequence encoding the Gal4 DNA binding domain. The recombinant plasmid was inserted into the human liver cDNA library cloned into pACT2 vector (Fused to a DNA sequence encoding a Gal4 activation domain) were co-transformed into yeast AH109. ECRG 1 interacts with the protein encoded by the corresponding human liver cDNA fragment to activate reporter gene expression. After excluding false positives, library cDNAs in positive clones were sequenced. Homology search searches for the same or similar sequences in GenBank. The results were co-transformed to obtain about 3 × 10 6 transformants, 23 clones have the reporter gene expression. After excluding the false positives, two positive clones were obtained, which code for myc interactingznfingerprotein 1 and actin bindingprotein 2 80, respectively. Conclusion ECRG 1 gene can specifically bind to Miz 1 and FLNA in yeast, suggesting that ECRG 1 may be involved in the regulation of cell cycle by interacting with MIZ 1 and FLNA.