腺病毒介导HSV-2 gD基因修饰的树突状细胞疫苗制备

发布时间：2018-02-11 16:28

  本文关键词： 单纯疱疹病毒 树突状细胞 疫苗　出处：《医学研究生学报》2015年01期 　论文类型：期刊论文

【摘要】：目的生殖器疱疹尚无彻底治愈的方法。研制有效的生殖器疱疹病毒疫苗是预防和治疗HSV-2感染的关键,文中探讨制备腺病毒介导的HSV-2 g D基因修饰的树突状细胞(dendritic cell,DC)疫苗的可行性。方法将HSV-2 g D蛋白基因克隆到质粒载体Shuttle-2,重组质粒酶切鉴定、测序鉴定后构建重组腺病毒p Adeno-HSV-2 g D。分离小鼠骨髓DC细胞,p Adeno-HSV-2 g D转染DC细胞后用流式细胞术检测DC表型,用免疫组化法、RT-PCR、SDS-PAGE和Western blot方法检测HSV-2 g D的表达情况。结果以HSV-2病毒DNA为模板,采用g D基因引物扩增出相应的目的片段。琼脂糖凝胶电泳显示,PCR产物g D基因同预期的大小一致(1182 bp)。p Adeno-g D DNA用脂质体法转染293细胞10 d,反复冻融获得p AdenoHSV-2 g D重组腺病毒,活性为4×1010IU/m L。流式细胞仪检测结果显示:成熟DC和腺病毒感染后DC,CD40含量为(74.2±3.9)%、(81.3±3.1)%,CD80含量为(73.9±4.1)%、(80.4±2.9)%,CD86含量为(76.1±5.5)%、(83.7±3.9)%,均较不成熟DC的CD40、CD80、CD86含量[(9.7±0.5)%、(7.5±1.2)%、(5.2±1.1)%]升高(P0.01)。p Adeno-HSV-2 g D-DC和细胞因子刺激诱导成熟的DC细胞表面分子表达差异无统计学意义(P0.05)。RT-PCR、免疫组织化学方法证明HSV-2 g D能在DC细胞内表达,表达产物的SDS-PAGE和Western blot分析发现,在相对分子质量为43000处有外源蛋白表达,与预期蛋白条带一致。结论成功制备了p Adeno-HSV-2 g D-DC疫苗。
[Abstract]:Objective there is no complete cure for genital herpes. The key to prevent and treat HSV-2 infection is to develop an effective vaccine against genital herpes virus. To investigate the feasibility of the preparation of adenovirus-mediated dendritic cell dendritic cell dendritic cell vaccine mediated by adenovirus. Methods the HSV-2 G D gene was cloned into the plasmid vector Shuttle-2 and identified by restriction endonuclease digestion. The recombinant adenovirus p Adeno-HSV-2 g D was constructed after sequencing. The DC cells were isolated from mouse bone marrow DC cells and transfected with p Adeno-HSV-2 g D. The phenotypes of DC cells were detected by flow cytometry. Immunohistochemical method was used to detect the expression of HSV-2 g D by RT-PCR- SDS-PAGE and Western blot. Results the HSV-2 virus DNA was used as a template. The corresponding target fragment was amplified by using the primers of G D gene. Agarose gel electrophoresis showed that the G D gene of the PCR product was in accordance with the expected size of 1182 bp).p Adeno-g D DNA transfected into 293 cells for 10 days by liposome method, and the recombinant adenovirus of p AdenoHSV-2 g D was obtained by repeated freezing and thawing. The activity was 4 脳 10 ~ (10) IUM 路L ~ (-1). The results of flow cytometry showed that the content of CD40 in mature DC and adenovirus infected DC was 74.2 卤3.9 卤3.9 卤3.1%. The content of CD80 was 73.9 卤4.1 卤4.1%, and the content of CD86 was 76.1 卤5.55.5U / L, which was higher than that of immature DC's CD40CD80C (9.7 卤0.5 卤7.5 卤1.2 卤1.2 卤1.1%), and the level of Adeno-HSV-2 g D-DC and cytokine stimulated mature DC were higher than that of immature DC (9.7 卤0.5 卤7.5 卤1.2 卤1.1%). There was no significant difference in the expression of HSV-2 g D on the surface of DC cells. The expression of HSV-2 g D in DC cells was confirmed by immunohistochemical method. SDS-PAGE and Western blot analysis showed that exogenous protein was expressed at a molecular weight of 43000, which was consistent with the expected protein band. Conclusion the p#en3# g D-DC vaccine was successfully prepared.
【作者单位】： 广州军区广州总医院皮肤科;
【基金】：国家自然科学基金(81071401)
【分类号】：R392
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