中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (50): 9419-94.doi: 10.3969/j.issn.1673-8225.2011.50.027

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

重组骨形成蛋白4/7融合基因腺相关病毒载体的构建与鉴定

袁绍辉，刘  伟，吴滨奇，韩晰光，毕郑钢   

  1. 哈尔滨医科大学附属第一医院骨一科，黑龙江省哈尔滨市 150001
  • 收稿日期:2011-06-18 修回日期:2011-09-20 出版日期:2011-12-10 发布日期:2011-12-10
  • 通讯作者: 毕郑钢，哈尔滨医科大学附属第一医院骨一科，黑龙江省哈尔滨市 150001 yuansh1970@sina.com
  • 作者简介:袁绍辉☆，1970年生，男，吉林省辽源市人，汉族， 2005年吉林大学毕业，博士，主要从事创伤与骨显微外科研究。 yuansh1970@sina.com
  • 基金资助:

    课题受黑龙江省自然科学基金资助项目(D200876)和黑龙江省教育厅科学技术研究项目资助项目(11531149)资助。

Construction and identification of recombinant adeno-associated virus vector with human bone morphogenetic protein 4/7 fusion gene

Yuan Shao-hui, Liu Wei, Wu Bin-qi, Han Xi-guang, Bi Zheng-gang   

  1. First Department of Orthopedics, First Hospital of Harbin Medical University, Harbin  150001, Heilongjiang Province, China
  • Received:2011-06-18 Revised:2011-09-20 Online:2011-12-10 Published:2011-12-10
  • Contact: Bi Zheng-gang, First Department of Orthopedics, First Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China yuansh1970@sina.com
  • About author:Yuan Shao-hui☆, Ph.D., First Department of Orthopedics, First Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China yuansh1970@sina.com
  • Supported by:

    the Natural Science Foundation of Heilongjiang Province, No. D200876*; Science and Technology Research Project of Education Department of Heilongjiang Province, No. 11531149*

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摘要：

背景：有研究表明骨形成蛋白(bone morphogenetic protein，BMP)异源二聚体比同源二聚体的活性高20倍，但相关BMP 4/7融合基因的相关病毒载体的构建至今少有报道。
目的：构建重组人BMP-4/7 融合基因腺相关病毒(adeno-associated virus，AAV)载体并检测其表达。
方法：扩增BMP-4、7成熟肽基因；分别构建质粒pGEM-BMP-4和pGEM-BMP-7；采用DNA连接法获取BMP-4/7融合基因，克隆获得pGEM-BMP-4/7质粒，进行基因测序。AAV颗粒转染HEK293细胞，收获病毒载体AAV-BMP-4/7。用SDS-PAGE电泳检测BMP-4/7融合基因蛋白在大肠杆菌的表达。用不同感染复数的AAV-BMP-4/7转染骨髓间充质干细胞3 d，提取细胞总RNA和总蛋白，进行RT-PCR和ELISA法检测融合基因BMP-4/7在骨髓间充质干细胞中的表达。
结果与结论：PCR 电泳，酶切鉴定及测序结果表明，装入pSNAV质粒中的BMP-4/7 基因正确，pSNAV-BMP-4/7重组成功。RT-PCR检测到骨髓间充质干细胞BMP-4/7融合基因的转录条带，ELISA检测显示经AAV转染的骨髓间充质干细胞中BMP-4/7蛋白随着感染复数的增加表达逐渐增高(P < 0.01)。结果证实，实验成功构建重组骨形成蛋白4/7融合基因腺相关病毒载体。

关键词: 重组人骨形成蛋白, 融合基因, 腺相关病毒, 骨髓间充质干细胞, 骨组织工程

Abstract:

BACKGROUND: Studies have shown that the activity of bone morphogenetic protein (BMP) heterodimer is 20 times higher than that of BMP homodimer, but related BMP 4/7 fusion gene-associated virus vector has been rarely reported.
OBJECTIVE: To construct the recombinant adeno-associated virus vector with human BMP 4/7 fusion gene (AAV-BMP-4/7) and to detect its expression.
METHODS: Human BMP 4, 7 mature peptide gene was amplified. Plasmids pGEM-human BMP 4, 7 were constructed, respectively. BMP 4/7 fusion gene was harvested using DNA ligation and then cloned to harvest pGEM-BMP-4/7 plasmid for gene sequencing. HEK293 cells were transfected with adeno-associated virus (AAV) particles to harvest viral vector AAV-BMP-4/7. BMP-4/7 fusion gene protein expression in E. coli was detected by SDS-PAGE. AAV-BMP-4/7 of different multiplicities of infection (MOI) was used to transfect bone marrow stromal cells (BMSCs) for 3 days. Total cellular RNA and total cellular protein were extracted. BMP-4/7 fusion gene expression in BMSCs was detected by RT-PCR and ELISA.
RESULTS AND CONCLUSION: The constructed recombinant pSNAV-BMP-4/7 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequence in the recombinant pSNAV-BMP-4/7 was correct. Ttranscription bands of BMP-4/7 fusion gene in BMSCs were detected by RT-PCR. ELISA showed that BMP-4/7 protein in the AAV-transfected BMSCs was gradually increased with the increase in MOI (P < 0.01). These results showed that the AAV-BMP-4/7 was successfully constructed.

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