hTPO膜外区基因的克隆及其杆状病毒表达载体的构建

Cloning of Extracellular Domain Gene of Human Thyroid Peroxidase and Construction of Its Baculovirus Expression Vector

目的:探索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。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.coliDH10Bac大肠杆菌,获得重组hTPO杆状病毒表达载体(hTPO-Bacmid),每一步均以PCR及酶切、基因测序等方法鉴定其正确性。结果:与预期一致,PCR扩增hTPO膜外区基因的产物为一约2.5kb的条带;hTPO-pGEM3zf(+)经EcoRⅠ+HindⅢ、EcoRⅠ+SalⅠ双酶切后均获得2.5和3.2kb条带;hTPO-pFastBAC1以EcoRⅠ+HindⅢ双酶切得到2.5和4.8kb的带,均符合预期。分别以重组hTPO-pGEM3zf(+)质粒、hTPO-pFastBAC1质粒为模板进行PCR扩增鉴定,均得到约2.5kb的目的条带;对hTPO-Bacmid进行PCR鉴定,得到约4.8kb的片段。对hTPO-pFastBAC1上下游接口测序正确无误。结论:本研究成功克隆了hTPO膜外区基因,并完成了其杆状病毒表达载体hTPO-Bacmid的构建。

Objective： To explore the cloning of extracellular domain gene of human thyroid peroxidase （hTPO） and con- struct its baculovirus expression vector. Methods： The hTPO of extracellular domain gene was obtained by PCR method. The fragment was ligated with pGEM3zf （＋） vector and pFastBacl vector sequently to generate recombinant hTPO-pGEM3zf （＋） and hTPO-pFastBAC1. The hTPO-pFastBAC1 was used to transfect E.eoli DHIOBac to get the recombinant baculovirus ex- pression vector （hTPO-Baemid）. For each step, the accuracy was identified by methods of PCR, restriction endonuclease anal- ysis and DNA sequencing. Results： The PCR product of hTPO＇s extracellular domain gene was a fragment about 2.5 kb in length as predicted. After double restriction enzyme digestion of EcoR I ＋Hind III or EcoR I ＋Sal I, hTPO-pGEM3zf（＋） was divided into one 2.5 kb and a 3.2 kb band. One 2.5 kb and a 4.8 kb band were obtained, after double digestion of hT- PO-pFastBAC1 by EcoR I ＋HindⅢ. By method of PCR, using the recombinant hTPO-pGEM3zf （＋） and hTPO-pFastBAC1 as template, each of them produced a 2.5 kb fragment. The result of interface sequencing of hTPO-pFastBAC1 was also the same as predicted. Conclusion： The extracellular domain gene of hTPO was cloned successfully, and the construction of the recombinant baculovirus expression vector of hTPO was accomplished.