解剖学报

解剖学报 ›› 2016, Vol. 47 ›› Issue (6): 769-773.doi: 10.16098/j.issn.0529-1356.2016.06.008

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成骨细胞骨涎蛋白成熟肽修饰位点和进化踪迹分析

蔡敏1 林建立2* 侯建明2 汤发强3 晋龙4   

  1. 1.福建省立医院干部特诊二科，福建省临床老年病研究所; 2.内分泌科，内分泌重点实验室; 3.骨科; 4.病理科，福州 350001
  • 收稿日期:2016-08-22 修回日期:2016-09-07 出版日期:2016-12-06 发布日期:2016-12-06
  • 通讯作者: 林建立 E-mail:linjianli@hotmail.com
  • 基金资助:

    福建省自然科学基金;福建省自然科学基金;福建省内分泌临床重点专科项目资助

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

CAI Min1 LIN Jian-li 2* HOU Jian-ming2 TANG Fa-qiang3 JIN Long4   

  1. 1.Deptment of the Second Cadres Special Medicine, Fujian Provincial Clinical Senile Diseases Institute; 2.Endocrinology, Key Laboratory of Endocrinology; 3.Orthopedics; 4.Pathology, Fujian Provincial Hospital, Fuzhou 350001, China
  • Received:2016-08-22 Revised:2016-09-07 Online:2016-12-06 Published:2016-12-06
  • Contact: LIN Jian-li E-mail:linjianli@hotmail.com

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摘要：

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cDNA，以特异性引物扩增BSP成熟肽基因并克隆到pBluescript 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。

关键词: 骨涎蛋白, 配基结合口袋, 修饰位点, 进化踪迹分析, 细胞三维培养

Abstract:

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 cDNA fragment was obtained by RT-PCR with two gene specific primers. The segment was inserted into pBluescript 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. 900bp 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.

Key words: Bone sialoprotein, Ligand-binding pocket, Modification site, Evolutionary trace analysis, Three dimensional cell culture