登革病毒E蛋白区段基因克隆及高效表达

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。

High level expression of D2V E gene fragment in E. coli and analysis of expression product

Objective In order to obtain the product of D2V E gene fragment for establish ing the basis to analyse the epitopes and functions of T and B cells, the high level expression of the gene fragment in E. coli was studied. Methods The gene fragment coding for 308 468aa of envelope (D2V E) protein of Dengue 2 virus was cloned with PCR method from p30VD2 plasmid and inserted into pMY vector with P L promoter. The recombinant plasmid was transformed into E. coli cells and the expressed products were analyzed with SDS PAGE and Western blotting. Results High level expression was performed by truncating coding region for the hydrophobic carboxyterminus of the D2V E protein. The expressed product of the gene fragment could amount to 25.31% of the total bacterial protein. The product was in the form of inclusion bodies and could be solubilized in 4mol/L urea and served as antigen to coat microtitre plates for detection of anti DV antibodies. The results showed that the recombinant protein reacted with antibodies from mice immunized with DV 1 4 type. Conclusions This study suggests that the gene fragment lacking the sequences coding for the hy drophobic carboxyterminus of the D2V E protein be expressed efficiently in E. coli system and the expressed products react with antibodies from mice immunized with DV 1 4 type.