中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (18): 3331-3334.doi: 10.3969/j.issn.1673-8225.2010.18.024

• 器官移植基础实验 basic experiments of organ transplantation • 上一篇    下一篇

大鼠Delta1基因真核表达载体的构建与鉴定

郑  凯，谭建明，吴卫真，杨顺良   

  1. 解放军南京军区福州总医院全军器官移植中心，福建省福州市 350025
  • 出版日期:2010-04-30 发布日期:2010-04-30
  • 基金资助:

    福建省自然科学基金资助项目。

Construction and identification of eukaryotic expression vector of rat Delta1 gene

Zheng Kai, Tan Jian-ming, Wu Wei-zhen, Yang Shun-liang   

  1. Organ Transplant Institute, Fuzhou General Hospital of Nanjing Military Area Command of Chinese PLA, Fuzhou   350025, Fujian Province, China
  • Online:2010-04-30 Published:2010-04-30
  • Supported by:

    the Natural Science Foundation of Fujian Province*

PDF

432

被引次数

3

摘要：

背景：最近有数据表明，Notch信号通路在外周移植免疫应答反应中发挥重要的调节作用，可以促进调节性T的分化，诱导抗原特异性的免疫耐受。推测Notch/Notch 配体可能在MHC：TCR界面发挥作用。
目的：构建大鼠Delta1基因(Notch 配体)真核表达载体，并观察其在树突状细胞中的表达。
方法：应用RT-PCR方法从大鼠骨髓细胞中获得全长Delta1基因片段，并将此基因片段构建在pcDNA3.1(+)真核表达载体上。利用脂质体基因转染技术将含大鼠Delta1基因片段的真核表达载体pcDNA3.1(+)/Delta1转染入树突状细胞中，观察Delta1基因在树突状细胞中的表达。
结果与结论：双酶切鉴定结果显示，Delta1已成功构建在pcDNA3.1的HindIII和XbaI双酶切位点之间，任选一个酶切阳性克隆pcDNA3.1/Delta1送上海生工生物公司进行序列测定，测序结果与Genebank中Delta1基因序列完全一致，阅读框正确。转染Delta1基因的树突状细胞形态与其亲本细胞类似，蛋白免疫印迹法检测到细胞内Delta1的表达显著增高。实验成功利用基因转染方法将Delta1基因导入树突状细胞，构建了真核表达载体pcDNA3.1(+)/Delta1，并使之高效表达并分泌Delta1蛋白。

关键词: 树突状细胞, Notch信号, Delta1, 免疫耐受, 移植, 大鼠

Abstract:

BACKGROUND: Recent data suggested that Notch signal pathway plays important regulatory effects in peripheral transplantation immunological response, promotes differentiation of regulatory T cells, induces antigen specific immune tolerance. We proposed that Notch/Notch ligand may play important roles in MHC/TCR interface.
OBJECTIVE: To construct the eukaryotic expression vector of rat Delta1 gene (Notch ligand), and to examine its expression in dendritic cells.
METHODS: The complete encoding cDNA of rat-Delta1 was isolated from bone marrow cells by reverse transcription-polymerase chain reaction (RT-PCR) and this gene was recombined into pcDNA3.1(+) plasmid vector. pcDNA3.1/Delta1 plasmid was transfected into rat dendritic cells with lipofectamine gene transfection method.
RESULTS AND CONCLUSION: Double enzyme digestion detection demonstrated that Delta1 had been successfully constructed in HindIII and XbaI of pcDNA3.1. A positive clone pcDNA3.1/Delta1 was delivered to Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. for sequencing. Sequencing results were identical to Delta1 gene sequence in Genebank, with correct reading frame. The Delta1 gene-transfected dendritic cells showed similar morphology as their parent cells. Western blotting assay detected that Delta1 expression was significantly increased in cells. The eukaryotic expression vector pcDNA3.1/Delta1 was constructed, and significant increase of Delta1 expression was detected after transfection.

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