成骨细胞骨涎蛋白成熟肽修饰位点和进化踪迹分析

Analysis of bone sialoprotein and mature peptide modification sites and evolutionary trace

目的探讨成骨细胞中骨涎蛋白(BSP)的表达以及进化踪迹,并对成熟肽基因行序列分析,获得BSP修饰位点。方法体外培养成骨细胞,提取成骨细胞总RNA,反转录合成c DNA,以特异性引物扩增BSP成熟肽基因并克隆到p Bluescript KS载体,筛选阳性克隆进行序列测定。以成骨细胞的骨涎蛋白为种子序列,利用多种生物信息学工具进行细胞的骨涎蛋白的序列查找及其同源蛋白的搜索,并以此为基础对骨涎蛋白的进化踪迹位点及相关的功能位点进行比较研究。结果共得到具有完整结构域的同源蛋白序列72条,骨涎蛋白家族的BSP-前肽结构域具16个全家族保守残基,以及10个RGD结合域和BSP结构域的33个亚家族特异性残基。骨涎蛋RGD结构域的配基结合位点主要分布于结合口袋的中间区域。PCR扩增到一特异性的900bp片段,克隆后筛选出阳性克隆进行序列分析,测定结果与数据库的序列基本相同。结论成骨细胞中能表达并翻译为有活性的BSP,BSP成熟肽存在甲基和乙酰化修饰位点。

Objective To investigate the expression of osteoblasts bone sialoprotein（ BSP） and evolutionary trace.The mature peptide gene sequences were analyzed to obtain BSP modification sites. Methods Total RNA was isolated from cultured osteobalsts and desired c DNA fragment was obtained by RT-PCR with two gene specific primers. The segment was inserted into p Bluescript KS vector and the result plasmid was transformed into DH5α. The positive clone and sequence were performed. Using BSP as seed sequences,the searching of BSP gene sequence and its homologous protein with various bioinformatics tools were retrieved from NCBI database. The comparative studies were taken for BSP evolutionary trace sites and related functional sites. Results There was a complete structural domain of 72 homologous protein sequences,16 bone sialoprotein family BSP-propeptide domain,10 RGD binding domain and the BSP domain 33 subfamily specific residues. The ligand of bone sialoprotein RGD domain binding sites was mainly distributed in the middle region with pockets. 900 bp specific fragment was obtained. The sequence of the gene encoding BSP mature pepide coincided with that of the references published. Conclusion The gene encoding BSP mature peptide is obtained from osteoblasts that express and translate into active bone sialoprotein. There are methylation and acetylation sites existed on the BSP mature peptide.This result will help us to further investigate the expression and function of BSP.