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人甲状腺过氧化物酶膜外区基因的克隆及其杆状病毒表达载体的构建

刘金泉1,肖茜2,李宁3,方佩华3   

  1. 1. 天津医科大学总医院代谢病科
    2. 总医院核医学科
    3. 天津医科大学总医院
  • 收稿日期:2011-09-08 修回日期:2011-12-07 出版日期:2012-06-15 发布日期:2012-06-15
  • 通讯作者: 刘金泉

Cloning of the extracellular domain gene of human thyroid peroxidase and construction of it’s baculovirus expression vector

  • Received:2011-09-08 Revised:2011-12-07 Published:2012-06-15 Online:2012-06-15

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摘要： 摘要 目的：尝试克隆人甲状腺过氧化物酶（hTPO）膜外区基因并构建其杆状病毒表达载体，为其在昆虫细胞中的表达打好基础。方法：PCR扩增hTPO膜外区基因，并将其先后重组入pGEM3zf(+)质粒和pFastBac1质粒，以hTPO-pFastBAC1质粒转染E.coli DH10Bac大肠杆菌，获得重组hTPO杆状病毒表达载体（hTPO-Bacmid）,分别以多种方法鉴定其正确性。结果：经PCR及酶切、基因测序等方法证实得到的重组hTPO-pGEM3zf(+)质粒、hTPO-pFastBAC1质粒和hTPO-Bacmid均与预期相符。结论：成功的克隆了hTPO膜外区基因，并将其正确重组入pGEM3zf(+)质粒和pFastBac1质粒,后者经位点特异性转座整合至Bacmid，成功的构建了重组hTPO杆状病毒表达载体，为进一步实现hTPO在昆虫细胞中的真核表达作好了准备。

关键词: 人甲状腺过氧化物酶, 膜外区基因, 聚合酶链反应, 杆状病毒表达系统

Abstract: Abstract Objective: Try to clone the extracellular domain gene of human thyroid peroxidase(hTPO) and construct the baculovirus expression vector of it, so that we can carry out it’s expression in the baculovirus-insect cell expression system in the future. Method: Human TPO’s extracellular domain gene was obtained by PCR method. The fragment was ligated with pGEM3zf(+) vector and pFastBac1 vector sequently to generate recombinant hTPO-pGEM3zf(+) and hTPO-pFastBAC1. The hTPO-pFastBAC1 was used to transfect E.coli DH10Bac so as to get the recombinant baculovirus expression vector(hTPO-Bacmid). For each step, we identify the accuracy by several methods. Results: By methods of PCR, restriction endonuclease analysis and DNA sequencing, the recombinant hTPO-pGEM3zf(+),hTPO-pFastBAC1 and hTPO-Bacmid was confirmed the same as predicted. Conclusion: We have cloned hTPO’s extracellular domain gene successfully, and then generated recombinant hTPO-pGEM3zf(+) and hTPO-pFastBAC1 accurately. The latter one achieved recombination with Bacmid by site-specific transposition. So the recombinant baculovirus expression vector of hTPO was obtained. This work has established a basis for hTPO gene’s eukaryotic expression in insect cells.

Key words: human thyroid peroxidase, extracellular domain gene, PCR, baculovirus expression vector system