牛SRY蛋白表达纯化与体外功能鉴定

Expression and purification of bovine SRY protein and its functional characterization in vitro

利用PCR技术从北京黑白花奶牛(Bostaurus)的基因组DNA中克隆了SRY(Sex-determiningregionontheYchromosome)基因编码区全长序列。序列分析表明牛SRY基因的HMG区(Highmobilitygroup)呈现高度的保守性,与人、小鼠、猪等的相似性达到70%。将SRY基因与pET-28a(+)载体相连,构建表达载体pET-28a/SRY;把该表达载体转入大肠杆菌BL21(DE3),以IPTG诱导30℃诱导4h,SRY蛋白可高效表达,表达产物占总蛋白量的26%。对表达产物进行了Western-blotting检测,并采用亲和层析技术获得了高纯度的牛SRY蛋白。通过PCR技术分别获得牛、人、鼠的苗勒氏管抑制物(MullerianInhibitingsubstances,MIS)启动子,凝胶阻滞试验证明,牛SRY蛋白可与人及牛的MIS启动子结合,但与鼠的Mis启动子不发生相互作用。

The SR Y gene was cloned from the bovine Bos taurus genome. The total coding region is 687 bp, encoding a peptide with 229 amino acid residues. Compared with the SRY genes from other species, including human, mouse and pig, the amino acid sequence at the HMG region is highly conserved with 70% similarity. The bovine SRY gene was cloned to EcoR I and Sal I sites of pET-28a （ ＋ ） to construct an expression plasmid pET-28a-SRY. The expression plasmid was transformed to E. coli BL21 （DE3）, and induced by IPTG. SDS-PAGE analysis confirmed that the bovine SRY peptide was highly expressed after 4h induction at 30℃. The product of expression was identified by Western-blotting and the SRY protein was purified to homogeneity. Functional analysis using gel-shift experiments confirmed that bovine SRY protein could bind with the promoter of Mullerian Inhibiting Substances （MIS） from bovine and human. In contrast, no interaction was found between bovine SRY protein and the promoter region of MIS from mice .