siat7e基因和st3gal Ⅰ基因双表达载体的构建及其初步应用

The Construction and Preliminary Application of Double-gene Express Vector that Express Siat7e Gene and St3gal Ⅰ Gene Simultaneously

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1011 bp的唾液酸转移酶(siat7e基因),并从鸡基因组中扩增出全长为1029 bp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。本研究为最终构建能够适应悬浮培养,并且对禽流感病毒更为易感的MDCK细胞系奠定了基础。

The complete gene of sialyl transferase （siat7e gene） was amplified from human genome by polymerase chain reaction（PCR）, and the complete gene of α- 2,3 -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 siatTe gene was ligated with express vector pReceiver by BamH I/Psi I 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. This study establishing foundation for the final construction of a MDCK cell line that adapt to suspention culture and more susceptible to avian influenza virus.