【摘要】 目的克隆大鼠DesertHedgehog(DHH)基因,构建其真核表达载体并转染PT67细胞;同时制备DHHcRNA正义及反义探针用于检测其在细胞中的表达。方法提取SD大鼠睾丸总RNA,RT-PCR法扩增DHH-cDNA片段,连接于pGEM-TEasy载体,经测序后构建真核表达载体pLXSN/DHH并转染PT67细胞;重组质粒经限制性内切酶NotI和NcoI酶切、回收后,进行转录标记反应,原位杂交检测DHH在PT67细胞中的表达。结果RT-PCR扩增得到1220bp的片段;成功构建了真核表达载体pLXSN/DHH;制备DHH正义及反义探针浓度分别为150mg/L和80mg/L;DHH在PT67细胞中有表达。结论克隆的DHH基因与大鼠Sertoli细胞的DHH基因相同,成功标记了特异、敏感的DHHcRNA探针,转染的DHH基因能够在PT67细胞中表达。更多还原

【Abstract】 Objective In order to investigate the biological roles of Desert Hedgehog (DHH) expressed by Sertoli cells, we planed to clone the gene of DHH from rat, to construct its eukaryiotic expression vector, meanwhile, to label DHH sense and antisense probes and to detect the expression of DHH gene in PT67 cells. Methods Total cellular RNA of rat’s testes was abstracted and the cDNA fragment was amplified by RT-PCR. The fragment cDNA was ligated to pGEM-T easy vector by T4 DNA ligationase and sent to test sequence, and its sequence was analyzed. Plasmid pGEM-T easy/DHH and pLXSN plasmids were extracted, digested with EcoR I and BamH I, then ligated by T4 DNA ligationase. The eukaryiotic expression vector—pLXSN-DHH was constructed and then transfected into PT67 cells. The pGEM-T easy-DHH was digested by Not I and Nco I restriction enzymes and DHH cRNA probes were labeled by Dig-cRNA probe labeling kit. The expression of DHH was examined by in suit hybridization. Results The cDNA fragment of 1?220?bp was amplified by RT-PCR. An eukaryiotic expression vector, pLXSN-DHH, was successfully constructed. Anti-sense and sense probes were respectively 150?mg/L and 80?mg/L, and DHH gene was expressed in PT67 cells. Conclusion The DHH gene cloned in present study is same as the DHH gene in Sertoli cells. DHH probes were labeled successfully. The newly constructed vector, pLXSN-DHH, can be transfected into and expressed in PT67 cells.更多还原