骨肉瘤细胞中SORBS1的表达和定位

山东大学学报(医学版) ›› 2012, Vol. 50 ›› Issue (2): 21-.

骨肉瘤细胞中SORBS1的表达和定位

沈涛1，代炜2，李妍3，许晓军1，巴根1，付勤1

1.中国医科大学附属盛京医院骨科, 沈阳 110004；
2.中国医科大学附属口腔医院口腔颌面外科教研室口腔颌面头颈外科, 沈阳 110002；
3.中国医科大学基础医学院细胞生物学教研室，细胞生物学卫生部重点实验室，沈阳 110001

收稿日期:2011-09-19 出版日期:2012-02-10 发布日期:2012-02-10
通讯作者: 付勤(1958- )，主任医师，博士生导师，主要从事肿瘤转录调控的研究。 E-mail: cmuqinfu@gmail.com
作者简介:沈涛(1979- )，男，主治医师，博士研究生,主要从事肿瘤转录调控的研究。
基金资助:
国家自然科学基金资助项目(No.30800415和81070688)

Expression and localization of SORBS1 in osteosarcoma cells

SHEN Tao1, DAI Wei2, LI Yan3, XU Xiao-jun1, BA Gen1, FU Qin1

1. Department of Orthopedic Surgery, Shengjing Hospital of China Medical University, Shenyang 110004, China;
2. Department of Oral and Maxillofacial Surgery, The Affiliated Stomatological Hospital, China Medical University,
Shenyang 110002, China; 3. Department of Cell Biology, The Key Laboratory of Cell Biology of
Ministry of Education and Public Health of China, College of Basic Medical Sciences,
China Medical University, Shenyang 110001, China

Received:2011-09-19 Online:2012-02-10 Published:2012-02-10

摘要/Abstract

摘要：

目的构建人SORBS1真核表达载体并检测在骨肉瘤细胞内的表达及定位。方法以GST-hSORBS1为模板，利用PCR扩增hSORBS1基因cDNA全长，并将其克隆至3*Flag标签的真核表达载体中。进一步将构建的重组载体进行酶切和测序鉴定，并转染到骨肉瘤细胞MG-63中，提取蛋白进行Western blot检测。同时利用共聚焦激光显微镜观察3*Flag-hSORBS1在MG-63细胞中的定位，使用免疫沉淀的方法纯化hSORBS1蛋白。结果 hSORBS1基因cDNA全长成功构建到真核表达载体3*Flag中，Western blot检测到3*Flag-hSORBS1融合蛋白表达, 且在骨肉瘤细胞MG-63中主要定位于细胞周边, 并成功纯化hSORBS1蛋白。结论成功构建3*Flag-hSORBS1真核表达质粒，同时鉴定其融合蛋白的表达，并纯化hSORBS1蛋白。3*Flag-hSORBS1蛋白主要定位在细胞周边。

关键词: hSORBS1；骨肉瘤；真核表达载体；融合蛋白；免疫沉淀

Abstract:

Objective To construct an eukaryotic vector of human sorbin and SH3 domain containing 1 (hSORBS1) gene and identify the expression and localization of hSORBS1 in osteosarcoma MG-63 cells. Methods Using GST-hSORBS1 as a template, we obtained human SORBS1 coding sequence by polymerase chain reaction (PCR) amplification and cloned it into the eukaryotic vector 3*Flag. The insert was identified by restriction enzyme digestion and DNA sequencing. 3*Flag-hSORBS1 was transfected into osteosarcoma MG-63 cells. The expression of the recombinant plasmid in MG-63 cells was detected by Western blot. The localization of 3*Flag-hSORBS1 in MG-63 cells was observed with laser scanning confocal microscopy. The hSORBS1 fusion proteins were purified by immunoprecipitation assay. Results hSORBS1 was constructed into the eukaryotic vector 3*Flag successfully. The expression of 3*Flag- hSORBS1 fusion protein was detected by Western blot, which localization was at the periphery of MG-63 cells. Conclusion The eukaryotic expression plasmid of hSORBS1 is successfully constructed. The expression of fusion proteins is identified. 3*Flag-hSORBS1 fusion protein is localized at the cell periphery.

Key words: hSORBS1; Osteosarcoma; Eukaryotic expression plasmid; Fusion protein;Immunoprecipitation

中图分类号:

Q291

沈涛1，代炜2，李妍3，许晓军1，巴根1，付勤1. 骨肉瘤细胞中SORBS1的表达和定位[J]. 山东大学学报(医学版), 2012, 50(2): 21-.

SHEN Tao1, DAI Wei2, LI Yan3, XU Xiao-jun1, BA Gen1, FU Qin1. Expression and localization of SORBS1 in osteosarcoma cells[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2012, 50(2): 21-.

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