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目的进行登革病毒Ｅ蛋白区段的高效表达，制备抗原，为分析Ｔ、Ｂ细胞表位及免疫功能的研究奠定基础。方法以含有Ｄ２ＶＥ区的质粒ｐ３０．ＶＤ２为模板，ＰＣＲ扩增了编码Ｄ２ＶＥ蛋白３０８～４６８ａａ区段的基因片段，以ｐＭＹ为表达载体，构建了表达质粒ｐＤＥ１，并用酶切法使之缺失跨膜区疏水ａａ较多的编码序列，又构建了表达质粒ｐＤＥ２，分别转化大肠杆菌，获得了不同的工程菌株。经诱导培养，ＳＤＳ－ＰＡＧＥ，免疫印迹及酶联检测分析表达产物。结果含ｐＤＥ１质粒工程株只能表达微量的特异性蛋白；而含ｐＤＥ２质粒的工程菌株则能高效表达Ｄ２Ｖ特异性Ｅ蛋白，重组表达产物占菌体蛋白的２５．３１％。用４ｍｏｌ／Ｌ尿素溶解包涵体，上清中Ｅ蛋白纯度可达８０％。以粗提物包板进行酶联检测，结果表明，该重组Ｅ蛋白具有ＤＶ４个型交叉抗原的特性。结论缺失Ｃ末端疏水区编码序列的Ｄ２ＶＥ基因片段，可在Ｅ．ｃｏｌｉ中获得高效表达，其表达产物具有ＤＶ属特异性。
Objective To carry out high efficient expression of dengue virus E protein segment and preparation of antigens for the study of T, B cell epitopes and immune function. Methods Plasmid p30 containing the D2VE region. VD2 as a template, the gene fragment coding for the segment 308 to 468aa of D2VE protein was amplified by PCR. The expression plasmid pDE1 was constructed by using pMY as the expression vector, and the coding sequence of pDE1 was deleted by enzyme digestion. Also constructed the expression plasmid pDE2, were transformed into E. coli, obtained different engineering strains. The expression products were analyzed by induction culture, SDS-PAGE, Western blot and enzyme-linked assay. Results The engineering strain containing pDE1 plasmid could express only trace amount of specific protein. However, the engineering strain containing pDE2 plasmid could express D2V - specific E protein efficiently, and the recombinant product expressed 25.31% of the total bacterial protein. Inclusion bodies were solubilized with 4 mol / L urea, and the purity of E protein in the supernatant was up to 80%. The results of enzyme-linked immunosorbent assay (ELISA) showed that the recombinant E protein has the characteristics of DV4 cross-linked antigen. Conclusion The D2VE gene fragment lacking the C-terminal hydrophobic region coding sequence can be found in E. coli. Highly expressed in E. coli, and its expression product has the specificity of DV genus.