人骨唾液蛋白基因的克隆及其在毕赤酵母中的表达

Cloning and Expression of Human Bone Sialoprotein in Pichia pastoris GS115

利用RT PCR技术 ,从人骨膜组织总RNA中扩增长度为 1kb的人骨唾液蛋白 (bonesialoprotein ,BSP)基因编码区序列 ,克隆至pBluescriptⅡ ,进行双链核苷酸序列测定 .构建人BSP毕赤酵母表达质粒pPICZaA hBSP并导入毕赤酵母PichiapastorisGS1 1 5 .SDS PAGE和Western印迹检测rhBSP的分泌表达 .采用镍离子亲合层析纯化rhBSP并检测生物学活性 .结果表明 ,克隆片段与基因库所登录的序列相一致 ,分泌表达的rhBSP His融合蛋白分子量约为 6 7kD ,占培养上清总蛋白的 80 % ,浓度约为 0 2 4 8g L 。

One kb DNA fragment encoding for human bone sialoprotein(BSP) cDNA was amplified with human periosteum total RNA as template and cloned to pBluescript II. The nucleotide sequence analysis was performed. The expression vector pPICZaA hBSP gene was constructed, linearized and introduced to Pichia pastoris GS115 by electroporation. The expression of rhBSP was detected by SDS PAGE and Western blotting. The rhBSP was purified by Ni 2+ NTA column and its bioactivity was detected. It showed that the sequence of cloned DNA fragment was as same as the human BSP cDNA sequence in GenBank. The expressed product of rhBSP His was 67 kD approximately with a concentration of about 0.248 g/L (～80% of total protein in the supernatant). The purified rhBSP had the hydroxyapatite seed growth inhibition.