【摘要】 目的:构建经糖蛋白gD信号肽修饰的单纯疱疹病毒2型(HSV-2)糖蛋白gD、gB、gC和早期表达蛋白ICP27的多表位复合DNA疫苗。方法:国内HSV-2野生株经测序与PCR鉴定后利用PCR技术从其基因组中扩增出HSV-2 ICP27 377-459、gD146-179、gD223-306、gB529-606、gC247-282、gD1-776个基因片段,PCR鉴定后按先后顺序,采用多基因片段一步酶切连接法获得HV融合基因片段,经pGEMT载体克隆后定向插入真核表达质粒pcDNA3.1载体中,构建出重组真核表达质粒HV-pcDNA3.1,并对其进行酶切分析及测序鉴定。PCR扩增gD146-179信号肽片段,进一步酶切连接构建重组质粒gDs-HV-his-pcDNA3.1,并对其进行测序鉴定。结果:酶切重组质粒HV-pcDNA3.1,电泳可见两条带,分别为融合基因HV(1.3kb)和线性质粒pcDNA3.1(5.4kb)。测序结果表明,克隆基因插入方向正确,重组质粒gDs-HV-his-pcDNA3.1与GenBank HSV-2中相关序列同源性达99.9%。结论:成功构建了经糖蛋白gD信号肽修饰的HSV-2多表位复合DNA疫苗。更多还原

【Abstract】 Objective: To construct the multigenic DNA vaccine modified by signal peptide of glycoprotein D (gD), glycoprotein B (gB), glycoprotein C (gC), infectious cell protein 27(ICP 27). Methods: HSV-2 ICP27gene sequences (377-459, gD146-179, gD223-306, gB529-606, gC247-282, gD1-77) obtained from domestic HSV-2 wild strain by PCR amplification. Multiple DNA fragments were cloned into pGEMT vector by one step quickly cloning method, and the products of the fused aim gene HV were identified. HV was then directly cloned into eukaryotic expression vector pcDNA3.1, and got recombinant vector HV-pcDNA3.1 that was comfirmed by the restriction endonuclease and DNA sequencing. Signal peptide of gD was obtained by PCR amplification and got recombinant vector gDs-HV-his-pcDNA3.1 by endonuclease. Results: Restriction endonuclease digestion analysis of the recombinant vector HV-pcDNA3.1 showed two bands, one was fusion gene HV(1 300 bp), the other was linear pcDNA3.1(5 400 bp) that were correspondent with the aim gene fragments. DNA sequence analysis showed the orientation was correct and the rate of homology was 99.9% compared with HSV-2 DNA sequence from GenBank. Conclusion: Multigenic DNA vaccine modified by signal peptide of gD against herpes simplex virus type 2 has been successfully constructed.更多还原