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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。
Objective To clone and express the E protein gene of Dengue Ⅱ virus Tr1751 and construct the eukaryotic expression plasmid, which provided the conditions for its subsequent study on the structure and function. Methods E gene primers of DEN2 (Tr1751 strain) were synthesized. The gene fragment containing the signal peptide E protein was amplified by RT-PCR and ligated with eukaryotic vector p IRES-hr GFP-1α to obtain recombinant plasmid p IRES -hr GFP-1α-E, and then identified by PCR, double digestion and sequencing. Results The 1 527 bp E protein gene fragment was obtained by RT-PCR. Restriction analysis and sequencing showed that the recombinant plasmid contained the gene of E protein in DEN2 virus. The similarity of the nucleotide sequence and the deduced amino acid sequence was 99% after the recombinant plasmid was sequenced and compared with the known Tr1751 strain in Gen Bank. Conclusion The eukaryotic plasmid containing E protein gene of full-length DEN2 was successfully constructed and laid the foundation for the study of dengue virus nucleic acid vaccine.