摘要
目的构建单纯疱疹病毒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，探讨其诱导机体免疫应答的能力。方法将HSV-2野生株经PCR鉴定后，利用PCR技术从其基因组扩增出HSV-2 ICP27 377-459、gD146-179、gD223-306、gB529-606、gC247-282和gD1-77等6个基因片段。采用多基因片段一步酶切连接法获得构建的6个片段的复合DNA疫苗（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。10只C57／BL6小鼠均分为脂质体包裹pcDNA3．1空载体质粒对照组和脂质体包裹HV-pcDNA3．1质粒免疫组。ELISA检测小鼠血清HSV-2特异性IgG、IL-2和IFN-γ，乳酸脱氢酶法检测CTL功能。数据行t检验。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，与基因库HSV-2相关序列同源性达99．9％。与pcDNA3．1空载体质粒组相比，HV-pcDNA3．1免疫组小鼠血清中HSV-2特异性IgG（0．29±0．14比0．11±0．04，P〈0．05）、IL-2（1246．8±157．0比685．2±104．2，P〈0．05）和IFN-γ（1340．3±108．5比547．7±189．3，P〈0．05）表达明显增加，CTL活性增强（31．82±10．12比8．30±2．91，P〈0．05）。结论成功构建了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，能有效引起小鼠机体特异性体液免疫和细胞免疫应答。
Objective To construct the multiple epitope DNA vaccine of glycoprotein D （gD）, glycoprotein B （gB）, glycoprotein C （gC）, infectious cell protein 27（ICP27） against herpes simplex virus type 2 （HSV-2）, and investigate the immune responses induced by this vaccine. Methods Domestic HSV-2 wild strain was identified by polymerase chain reaction （PCR） amplification and HSV-2 ICP27 377-459, gD146-179, gD223-306, gB529-606, gC247-282, gD1-77 gene were amplified by PCR from the genome. Multiple DNA fragments were cloned into pGEMT vector by one step quickly cloning method, and the products were fused to eukaryotic expression vector pcDNA3.1 plasmid to obtain recombinant vector HV-pcDNA3.1 which was confirmed by the restriction endonuclease analysis and DNA sequencing. Five C57/BL6 mice were inoculated intramuscularly with the liposome-coated pcDNA3. 1 and liposome-coated HV-pcDNA3.1, respectively. The levels of HSV-2 specific IgG, interleukin （IL）-2 and interferon （IFN）-γ in serum were determined by enzyme-linked immunosorbent assay （ELISA）. The function of cytotoxicity T lymphocyte （CTL） was detected by lactate dehydrogenase （LDH） assay. The data were analyzed using t test. Results Restriction endonuclease analysis of the recombinant vector HV-pcDNA3.1 showed two bands, one was fusion gene HV（1. 3 kb）, the other was linear pcDNA3. 1 （5.4 kb）. DNA sequencing data showed correct orientation of the gene insertion and the homology was 99. 9% compared with HSV-2 in GenBank. HV-pcDNA3.1 significantly enhanced the levels of HSV-2 specific IgG （0.29 ± 0.14 vs 0. 11 ± 0. 04）, IL-2（1 246.8 ± 157.0 vs 685.2 ± 104.2）, IFN-γ （1 340.3 ± 108.5 vs 547.7 ± 189.3） and CTL （31.82 ± 10. 12 vs 8.30± 2.91） were all significantly increased in HV-pcDNA3. 1 inoculated group than those in pcDNA3.1 inoculated group （all P 〈 0.05）. Conclusions The multiple epitope DNA vaccine of gD, gB, gC, ICP27 has been successfully constructed, which could effectively induce spec
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2008年第2期65-69,共5页
Chinese Journal of Infectious Diseases
基金
上海市科委资助课题（024119011）
