酵母双杂交技术筛选与绵羊微管解聚蛋白相互作用的蛋白

采用TRIzol法提取绵羊卵巢组织总RNA,运用RT–PCR技术扩增绵羊微管解聚蛋白基因(STMN1)的编码区序列;通过同源重组技术构建重组诱饵表达载体pGBKT7–STMN1,鉴定该诱饵蛋白在酵母双杂交系统中的自激活活性、细胞毒性和表达情况;利用诱饵质粒pGBKT7–STMN1从绵羊卵巢组织细胞的酵母双杂交cDNA文...

Screening of the proteins interacting with sheep stathmin by the yeast two-hybrid technology

The total RNA was extracted from sheep ovarian tissue by TRIzol method, and the coding region sequence of STMN1 gene was amplified by RT-PCR. The recombinant bait expression vector pGBKT7-STMN1 was constructed by homologous recombination technology, and the self-activation activity, cytotoxicity and expression of the bait protein in yeast two-hybrid system were determined. Screening of the interacting host proteins was performed from the yeast two-hybrid cDNA library of sheep ovarian tissue cells with the bait plasmid pGBKT7-STMN1. The results showed that the plasmid pGBKT7-STMN1 was successfully constructed in the study, and had no self-activation activity and cytotoxicity to yeast cells, and could be normally expressed in yeast cells. Totally, 12 clones interacting with STMN1 were screened out, and the sequencing analysis and point-to-point revealed the two interacting proteins, CREB and FOXM1, which may be involved in the regulation of cell proliferation, cycle and apoptosis.