METH1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

目的通过观察血管生成抑制因子METH1的cDNA片段在酵母双杂交中的表达及检测其对报告基因有无激活作用,为进一步明确METH1抑制增生性瘢痕的分子机制奠定基础。方法采用酵母双杂交Gal4系统3,经PCR扩增子METH1的cDNA片段,分别克隆入pUC19质粒,经测序正确后,再分别亚克隆入酵母双杂交诱饵载体pGBKT7中。将重组质粒导入酵母菌AH109,检测其表达产物在酵母细胞中对报告基因的激活作用。结果成功获得METH1的cDNA片段,该片段所表达的蛋白对酵母菌AH109无毒性,且对报告基因无激活作用。结论血管生成抑制因子METH1蛋白活性区在酵母双杂交系统中的表达产物,可作为诱饵蛋白进行相互作用蛋白的筛选研究。

Expression of an angiogenesis inhibitor METH1 and assay of activation of reporter genes in yeast double-hybrid system

Objective This work is intended as a basis for further study of the molecular mechanism of METH1 inhibition of hypertrophic scar, through the expression of cDNA fragments of METH1 and assay of activation of reporter genes in yeast double-hybrid system. Methods cDNA fragments of METH1 were amplified by the polymerase chain reaction (PCR), and were subsequently cloned into pUC19. After verification by sequencing, they were subcloned into the vector pGBKT7 of the yeast double-hybrid system. These recombinant plasmids were then transfered into yeast fungus AH109, and their expression products were assayed for ability to activate the reporter genes. Results The cDNA fragments of METH1 were amplified successfully. The expression product of fragments were not toxic to AH109 and could not activate the reporter genes. Conclusion The expression product of the active fragment of METH1 can be used as a to search for interacting proteins in this yeast double-hybrid system.