单纯疱疹病毒1型糖蛋白D主要抗原表位区的表达、纯化及鉴定

发布时间：2018-01-10 14:09

本文关键词：单纯疱疹病毒1型糖蛋白D主要抗原表位区的表达、纯化及鉴定　出处：《基础医学与临床》2013年03期 　论文类型：期刊论文

【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。
[Abstract]:Objective to prokaryotic expression of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) main antigen epitope fusion protein, purification and identification of the methods by Protean software Lasergene7.0 for epitope prediction and analysis of HSV-1 gD amino acid sequence selected from the gD main antigenic region (gD MED) the region, synthetic cDNA sequences, construct the prokaryotic expression vector pET-GST-gD MED; the recombinant plasmid was transformed into Escherichia coli (E.coli) BL21 (DE3) pLysS, using isopropyl beta -D- thio galactopyranoside (IPTG) induced expression of gDMED fusion protein was purified by GST.Bind purification kit; Western blot gD MED the immunological activity of the fusion protein were analyzed. The results of HSV-1 gD in 266 ~ 394 region rich in epitope, synthetic cDNA sequences of the region, and successfully constructed the prokaryotic expression vector pET-GST-gD MED; gD MED fusion protein could be mainly Soluble expression, molecular mass is about 45 Ku, the purity can reach 95%; Western blot showed that gD MED fusion protein and serum in patients with HSV-1 infection occurred. Conclusion the specific binding was successfully expressed and purified with good antigenicity of HSV-1 gD fusion protein of MED, provides a theoretical basis for developing immune diagnosis kit and HSV gene engineering vaccine.

【作者单位】：北京大学人民医院检验科;北京大学工学院生物医学工程系;
【分类号】：R392.1
【正文快照】： 单纯疱疹病毒(herpes simplex virus type,HSV)属于疱疹病毒科α-亚科,是双链DNA病毒。感染HSV可导致疱疹性脑炎、生殖器疱疹和宫颈癌等多种疾病,是可导致胎儿先天性畸形和死亡的病原体之一[1]。目前,病毒培养被认为是HSV原发性感染早期诊断的“金标准”[2]。但是,在感染的恢

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