中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (24): 4402-4406.doi: 10.3969/j.issn.1673-8225.2010.24.008

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

人骨形态发生蛋白2和人成纤维细胞生长因子2双基因共表达腺病毒载体的构建及鉴定

郭伟韬，刘思景，王  辉，肖启贤，陈子秋，黄  云，王  斌   

  1. 广东医学院附属医院骨科，广东省湛江市  524001
  • 出版日期:2010-06-11 发布日期:2010-06-11
  • 作者简介:郭伟韬☆，男，1969年生，河南省禹州市人，汉族，2005年华中科技大学同济医学院毕业，博士，副主任医师，主要从事骨科基础与临床研究。广东医学院附属医院骨科，广东省湛江市 524001 Guoweitao2000@sina.com
  • 基金资助:

    广东省自然科学基金资助项目(8452402301001081)。

Construction and identification of recombinant adenovirus vector co-expressing human bone morphogenetic protein 2 and fibroblast growth factor 2 genes   

Guo Wei-tao, Liu Si-jing, Wang Hui, Xiao Qi-xian, Chen Zi-qiu, Huang Yun, Wang Bin   

  1. Department of Orthopaedics, Affiliated Hospital of Guandong Medical College, Zhanjiang  524001, Guangdong Province, China
  • Online:2010-06-11 Published:2010-06-11
  • About author:Guo Wei-tao☆, Doctor, Associate chief physician, Department of Orthopaedics, Affiliated Hospital of Guandong Medical College, Zhanjiang 524001, Guangdong Province, China Guoweitao2000@sina.com
  • Supported by:

    the Natural Science Foundation of Guangdong Province, No. 8452402301001081*

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被引次数

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摘要：

背景：内部核糖体进入位点序列载体能将上下游基因共同转录，故通过此载体可使连接在一起的人骨形态发生蛋白2和人成纤维细胞生长因子2同时高表达，可为骨缺损的治疗提供新方法。
目的：实验通过引入内部核糖体进入位点序列，采用基于attLXattR的λ噬茵体位点特异性重组系统的GatewayTM技术构建带有人骨形态发生蛋白2和人成纤维细胞生长因子2双基因重组腺病毒载体并进行鉴定。
方法：用聚合酶链反应方法扩增人骨形态发生蛋白2和人成纤维细胞生长因子2目的基因，将两个基因亚克隆至pIRES2-EGFP载体中，构建phBMP2-IRES-hFGF2载体，通过BP反应将hBMP2-IRES-hFGF2重组到载体pDONR221中，再通过LR反应将其转移到腺病毒载体pAd/CMV/V5-DEST中，线性化后转染293细胞进行包装获得重组腺病毒Ad-hBMP2-IRES-hFGF2。
结果与结论：phBMP2-IRES-hFGF2经酶切及测序鉴定正确，带hBMP2-IRES-hFGF2的载体在293细胞中包装成功，获得成熟的病毒颗粒，病毒滴度为2.82×1010 ifu/mL，证实成功构建了带有人骨形态发生蛋白2和人成纤维细胞生长因子2双基因重组腺病毒载体。

关键词: 人骨形态发生蛋白2, 人成纤维细胞生长因子2, 腺病毒载体, 内部核糖体进入位点序列, 转染, 基因治疗

Abstract:

BACKGROUND: The upstream and downstream genes can be co-transcribed by internal ribosome entry site (IRES) sequence, by which human bone morphogenetic protein 2 (hBMP2) and human fibroblast growth factor 2 (hFGF2) genes can be highly co-expressed, thus, it provides a new way for repairing bone defects.
OBJECTIVE: To construct the recombinant adenovirus vector co-expressing hBMP2 and hFGF2 depending on IRES sequence by λ phage-site specific recombination systems and to verify the correct recombination.
METHODS: BMP-2cDNA and FGF-2 cDNA were amplified by PCR, and were subcloned into the pIRES2-EGFP plasmid to obtain phBMP2-IRES-hFGF2 plasmid. It was subcloned into entry vector pDONR221 by BP reaction and subcloned into pAd/CMV/V5-DEST vector by LR reaction and linearization transfection 293 cells to obtain pAd-hBMP2-IRES-hFGF2.
RESULTS AND CONCLUSION: The target gene of hBMP2-IRES-hFGF2 was transferred into AD/CMV/V5-DEST vector correctly and it was confirmed by sequencing. The expression clone pAd-hBMP2-IRES-hFGF2 was packaged into maturated adenovirus successfully; the titer of Ad-hBMP2-IRES-hFGF2 was 2.82×1010 ifu/mL. The results demonstrated that adenovirus vector co-expressing hBMP2 and hFGF2 are constructed successfully. 

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