登革Ⅱ型病毒Tr1751株E蛋白基因的克隆与表达载体的构建

目的从登革Ⅱ病毒Tr1751株中克隆表达E蛋白的基因,构建了真核表达质粒,为其后继的关于结构和功能的研究提供了条件。方法合成DEN2(Tr1751株)的E蛋白基因引物,通过RT-PCR的方法扩增含有信号肽E蛋白的基因片段,并与真核载体p IRES-hr GFP-1α连接,得到重组的质粒p IRES-hr GFP-1α-E,再用PCR、双酶切和测序的方法进行鉴定。结果 RT-PCR得到的为1 527bp的E蛋白基因片段,酶切鉴定和测序显示重组的质粒含有DEN2病毒中的E蛋白的基因。用重组质粒测序后与Gen Bank中的已知Tr1751株进行相似性比对,核苷酸序列和推导的氨基酸序列同源性均为99%。结论成功的构建出含有全长DEN2的E蛋白基因的真核质粒,为登革病毒核酸疫苗的研究奠定了基础。

Cloning of envelope glycoprotein of Tr1751 strain of Dengue II virus and construction of a eukaryotic expression vector

Objective To construct a recombinant eukaryotic expression plasmid containing the E protein gene of dengue II virus for studying the structure and function of E protein and providing a potential source of developing dengue subunit vaccine. Methods E protein fragment was amplified by RT-PCR using primers and then cloned into pIRES-hrGFP-1α vector to construct the eukaryotic expression plasmid pIRES-hrGFP-la-E. Results The RT-PCR product was a gene fragment with 1 527bp ,which is consistent with the expected size. The recombinant expression plasmid was identified and verified as correct using colony PCR and double restriction enzyme digestion. After sequencing, the recombinant plasmid had 99% similarity to the nucleotide sequence and 99% similarity to the deduced amino acid sequence of the E protein gene（Tr1751） registered in GenBank. Conclusions A recombinant eukaryotic plasmid of E protein were constructed which including of Full length DEN2 E gene. Recombinant plasmid of E protein has laid the foundation for further development of vaccines against dengue.