登革2型病毒ZS01／01株E蛋白在真核细胞中的分泌表达研究

Secreted expression of dengue virus type 2 envelope glycoprotein in Eukaryotic cells

目的对登革2型病毒（DENV-2）ZSO1／01株E蛋白在哺乳动物细胞及昆虫细胞中的分泌表达进行研究。方法RT．PCR扩增DENV-2prM／E基因，通过融合PCR在prM基因前添加来自乙型脑炎病毒的信号肽序列，并将E基因羧基末端20％区域缺失或替换为乙型脑炎病毒SA14-2株E基因相应序列，将上述基因元件分别克隆人哺乳动物细胞表达载体pcDNA5／FRT及昆虫细胞表达载体pAcUW51-M中，将重组质粒转染293T细胞或Sf9细胞，利用间接免疫荧光（Immunofluoreseence assay，IFA）及Western Blot检测E蛋白的表达与分泌。结果各重组质粒分别转染293T细胞或Sit）细胞后，E蛋白在细胞内均有效表达，而仅有携带乙脑信号肽且缺失或替换E基因羧基末端20％区域的重组质粒转染293T细胞后，上清中可检测到明显的E蛋白分泌。结论信号肽及E基因羧基末端20％区域对登革病毒E蛋白的分泌至关重要，宿主细胞对其亦有一定影响。

Objective To secreted express envelope glycoprotein （E） of dengue virus type 2 extracellularly. Methods The entire prM/E gene was amplified by RT-PCR. An optimized signal sequence gene from Japanese encephalits virus （JEV, SAI4-14-2 strain） was introduced using fusion PCR. The impact of E protein transmembrane and cytoplasmatic domains was compared by amplifying prM and E with full length of E gene, with 20% truncation of the E gene at 3＇ terminus and one chimeric gene, which was generated by replacing the 3＇ terminal 20% region of E gene with the corresponding sequence of JEV （ SA14- 14-2 strain）. The PCR segments were inserted into the Nhel and NotI sites of pcDNA5/FRT vector or into the NheI and Xhol sites of pAcUW51-M. Then they were transfected into 293T cells or Sf9 cells respectively. The expression and secretion of E protein were detected by immunofluorescence assay （IFA） and Western Blot. Results After transected into 293T cells or Sf9 cells, all constructs expressed E protein intracellularly indentified by IFA while only two plasmids could secret detectable E protein into tissue culture using Western Blot analysis. Conclusion Signal peptide as well as the transmembrane and cytoplasmatic domains is crucial for the secretion of dengue E protein.