Wistar大鼠SONIC HEDGEHOG基因的克隆和表达

Cloning and expression of N-terminal of SHH gene in Wistar rat

为了获得SHH蛋白以研究它在治疗神经变性疾病中的作用 ,本室提取不同胚龄Wistar胎鼠中的总RNA ,应用RT PCR技术获得SHH N的cDNA ,重组于pGEM T载体 ,经测序鉴定后 ,构建原核表达质粒并获得表达菌株Q15 SHH N ,然后诱导SHH蛋白的表达。结果发现 ,在E11.5 E2 0 .5胎鼠的脊索中能克隆到 6 30bp的cDNA片段 ,并且随胚龄增加 ,此片段的表达量逐渐减少 ;测序结果显示 ,此片段及其所编码的氨基酸序列同数据库中人、小鼠和SD大鼠的SHH N的序列有较高同源性 ;表达菌株经诱导后产生约 2 1kD的融合蛋白。提示SHH N是一种发育相关基因 ,在不同种属中的同源性较高 。

To obtain sonic hedgehog (SHH) and to investigate its function in neurodegenerative disorders, reverse transcription-polymerase chain reaction (RT-PCR) methods were used to clone cDNA fragment of N terminal of SHH(SHH-N) gene from different tissues of Wistar embryonic rats at various stages. The acquired cDNA was subcloned into pGEM-T/pQE30 vectors and recombinants were then identified by sequencing. The expression of SHH-N protein was then induced by isopropyl thiogalactoside (IPTG). It had been found that the gene sequence showed some nuances as compared with that submitted to the PUBMED databank and the rat SHH-N protein of which the molecular weight is 21ku was expressed after induction. The results suggest that: SHH-N is a development-related gene and it is highly homologous among different species and could be normally expressed in prokaryotic system.