登革Ⅱ型病毒E蛋白Ⅰ、Ⅱ结构域在大肠杆菌中的表达及免疫反应性鉴定

摘要

目的 在大肠杆菌中表达登革Ⅱ型病毒包膜糖蛋白(E)的第一,二结构域(DⅠ/Ⅱ)并进行免疫反应性鉴定.方法 登革II型病毒接种乳鼠,提取总RNA,经RT-PCR扩增目的 基因片段.双酶切PCR纯化产物和质粒pET-28a(+)并连接构建重组质粒pET28a- DV2-E-D1&2.筛选阳性菌落,提取质粒并转化感受态细胞,IPTG诱导表达目的 蛋白并进行SDS-PAGE分析,Western blot 鉴定.结果 RT-PCR扩增得到约900 bp的目的 基因片段,双酶切及测序分析表明,插入序列正确且开放读码框正确.重组菌诱导后在37 kD位置有明显的诱导后表达带,其大小与预测值一致.经12%SDS-PAGE分析,显示重组蛋白主要以包涵体形式存在于沉淀中;Western blot结果 显示重组蛋白与His·Tag单抗和登革病毒单抗(D1-11)均发生反应.结论 成功地在大肠杆菌中表达登革II型病毒包膜糖蛋白(E)的DⅠ/Ⅱ,免疫反应性鉴定结果 表明其具有良好的免疫反应性.%In order to express the domain (D I, Ⅱ ) of DENV Ⅱ envelope protein in Escherichia coli, obtain the purified recombinant protein and identify its immunoreactivity, suckling mice were inoculated with live DENV II in the brain. The total RNA was extracted from the brain of the infected mice, and the envelope protein DNA fragment was amplified by RT-PCR and ligated into pET-28a to construct expression plasmid pET-28a ( + )-DV2-E-Dl&-2. The recombinant plasmid was transformed into E. Coli BL2KDE3) and induced by IPTG, and the expressed products were analyzed by SDS-PAGE and Western blot. After RT-PCR amplification, a specific DNA fragment of about 900 bp was obtained and ligated into pET-28a to construct the expression plasmid pET-28a( + )-DV2-E-Dl&-2. After induction with IPTG, a specific protein with a relative molecular mass of 37000 was obtained . After the recombinant strains lysed by ortexing and sonicating at A °C, the supernatant and precipitation were taken by 12% SDS-PAGE analysis, show recombinant protein mainly exists in the form of inclusion body in precipitation. Western blotting demostrated the reactivity of the recombinant protein with his. Tag McAb and DENV (Type I-IV) McAb. In condusion, the recombinant plasmid can be highly expressed in E. Coli BL21(DE3) in a soluble form and the recombinant protein can react with DENV (Type I-IV) McAb.