摘要
目的原核表达单纯疱疹病毒1型(HSV-1)糖蛋白D(gD)主要抗原表位区,纯化融合表达蛋白并对其进行鉴定。方法用Lasergene7.0中Protean软件对HSV-1 gD氨基酸序列进行抗原表位预测和分析,筛选出gD主要抗原表位区(gD MED),人工合成该区域cDNA序列,构建原核表达载体pET-GST-gD MED;将重组质粒转化大肠杆菌(E.coli)BL21(DE3)pLysS,用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,通过GST.Bind纯化试剂盒对gDMED融合蛋白进行纯化;Western blot对gD MED融合蛋白的免疫活性进行分析。结果 HSV-1 gD在266～394区域富含抗原表位,人工合成了该区域的cDNA序列,并成功构建了原核表达载体pET-GST-gD MED;gD MED融合蛋白主要以可溶形式表达,分子质量约为45 ku,纯度能达95%;Western blot结果显示,gD MED融合蛋白能与感染HSV-1患者的血清发生特异结合。结论成功表达和纯化了具有良好抗原性的HSV-1 gD MED融合蛋白,为HSV免疫诊断试剂盒和基因工程疫苗的研制提供了依据。
Objective： To express the major epitopes domain of herpes simplex virus type 1 （HSV-1） glycoprotein D （gD） in prokaryotic expression system. The fusion protein was purified and its immunogenicity was studied. Methods： The potential epitopes of HSV-1 gD were predicted using the software Protean of Lasergen 7.0. Then gD major epitopes domain （gD MED） was chosen based on the prediction results, whose cDNA sequence was synthesized and inserted into the prokaryotic expression vector pET-GST. The recombinant gD MED protein was expressed in E.coli BL21（DE3） pLysS with the induction of Isopropyl-β-D-1-Thiogalactopyrano -side （IPTG） and purified with GST？Bind purification kit. The immunological activity was analyzed by western blot. Results： HSV-1 gD MED was located at 266~394 region according to prediction results, whose cDNA sequence was synthesized and its prokaryotic expression vector pET-GST-gD MED was constructed successfully. The fusion gD MED protein could be expressed in soluble form, and its molecular weight （approximately 45 ku） was very close to predicted value. After purification, the purity quotient of the recombinant protein was over 95%. Western blot analysis revealed that the recombinant protein had immunological activity. Conclusions： HSV-1 gD MED fusion protein with immunological activity was expressed and purified, which would be helpful to prepare HSV immunologic diagnosis kit and genetically engineering vaccine.
出处
《基础医学与临床》
CSCD
北大核心
2013年第3期350-355,共6页
Basic and Clinical Medicine
关键词
单纯疱疹病毒1型
糖蛋白D
主要抗原表位区
原核表达
herpes simplex virus type 1
glycoprotein D
major epitopes domain
prokaryotic expression
