| 摘要: |
| 应用聚合酶链式反应(PCR)技术,从人类基因组中扩增出全长为1011bp的唾液酸转移酶(siat7e基因),并从鸡基因组中扩增出全长为1029bp的β-半乳糖苷 α-2,3-唾液酸转移酶 I(ST3GAL I)基因。将它们分别克隆入pMD18-T载体中。对扩增的全长siat7e基因序列采用BamH I 和 PstⅠ双酶切后连接入 pReceiver 真核表达载体,构建pRE-SIAT 重组表达载体,进而,对扩增的全长st3gal I基因采用Sac I 和 Xho I双酶切,后连接入pRE-SIAT 重组表达载体,构建pRE-SIAT-ST3 重组真核表达载体。经限制性内切酶消化及DNA序列分析,证实构建的重组表达质粒是正确的。将构建的双基因表达载体转染MDCK细胞,利用荧光显微镜观察,可见细胞表达明显的绿色荧光,从而初步证实了外源基因在MDCK细胞中的正确表达。 |
| 关键词: siat7e基因 st3gal I基因 表达载体 MDCK细胞 |
| DOI： |
| 投稿时间：2012-08-14修订日期：2012-09-26 |
| 基金项目:国家自然科学基因项目(项目批准号31101817)和“863”项目课题“疫苗与诊断试剂创制及工艺创新”子课题：“重大动物疫病疫苗悬浮培养关键技术研究”(所属课题编号 2011AA10A213)。 |
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| the construction and preliminary application of double-gene express vector that express siat7e gene and st3gal I gene simultaneously |
| 陈小云 |
| (China institute of veterinary drug control) |
| Abstract: |
| The complete gene of sialyl transferase (siat7e gene) was amplified from human genome by polymerase chain reaction(PCR), and the complete gene of α2,3-galactosyltransferase Ⅰ（ST3GAL I) gene was amplified from chicken genome by PCR. Then the two genes were cloned into clone vector pMD18-T seperately. The complete siat7e gene was ligated with express vector pReceiver by BamH I / PstⅠ double digestion to construct the pRE-SIAT express vector. Then the complete st3gal I gene was ligated with express vector pRE-SIAT by Sac I/Xho I double digestion to construct the pRE-SIAT-ST3 express vector. Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid was correct．The double-gene express vector was transfected into MDCK cell. Detected by fluorescence microscope，the GFP was distributing in the transfected cell. So we can conclude that the foreign genes were express corrected in the MDCK cell. |
| Key words: siat7e gene st3gal I gene express vector MDCK cell |
