發(fā)布時間：2018-02-11 16:28

  本文關(guān)鍵詞： 單純皰疹病毒 樹突狀細(xì)胞 疫苗　出處：《醫(yī)學(xué)研究生學(xué)報》2015年01期 　論文類型：期刊論文

【摘要】：目的生殖器皰疹尚無徹底治愈的方法。研制有效的生殖器皰疹病毒疫苗是預(yù)防和治療HSV-2感染的關(guān)鍵,文中探討制備腺病毒介導(dǎo)的HSV-2 g D基因修飾的樹突狀細(xì)胞(dendritic cell,DC)疫苗的可行性。方法將HSV-2 g D蛋白基因克隆到質(zhì)粒載體Shuttle-2,重組質(zhì)粒酶切鑒定、測序鑒定后構(gòu)建重組腺病毒p Adeno-HSV-2 g D。分離小鼠骨髓DC細(xì)胞,p Adeno-HSV-2 g D轉(zhuǎn)染DC細(xì)胞后用流式細(xì)胞術(shù)檢測DC表型,用免疫組化法、RT-PCR、SDS-PAGE和Western blot方法檢測HSV-2 g D的表達(dá)情況。結(jié)果以HSV-2病毒DNA為模板,采用g D基因引物擴(kuò)增出相應(yīng)的目的片段。瓊脂糖凝膠電泳顯示,PCR產(chǎn)物g D基因同預(yù)期的大小一致(1182 bp)。p Adeno-g D DNA用脂質(zhì)體法轉(zhuǎn)染293細(xì)胞10 d,反復(fù)凍融獲得p AdenoHSV-2 g D重組腺病毒,活性為4×1010IU/m L。流式細(xì)胞儀檢測結(jié)果顯示:成熟DC和腺病毒感染后DC,CD40含量為(74.2±3.9)%、(81.3±3.1)%,CD80含量為(73.9±4.1)%、(80.4±2.9)%,CD86含量為(76.1±5.5)%、(83.7±3.9)%,均較不成熟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和細(xì)胞因子刺激誘導(dǎo)成熟的DC細(xì)胞表面分子表達(dá)差異無統(tǒng)計學(xué)意義(P0.05)。RT-PCR、免疫組織化學(xué)方法證明HSV-2 g D能在DC細(xì)胞內(nèi)表達(dá),表達(dá)產(chǎn)物的SDS-PAGE和Western blot分析發(fā)現(xiàn),在相對分子質(zhì)量為43000處有外源蛋白表達(dá),與預(yù)期蛋白條帶一致。結(jié)論成功制備了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 鹵3.9 鹵3.9 鹵3.1%. The content of CD80 was 73.9 鹵4.1 鹵4.1%, and the content of CD86 was 76.1 鹵5.55.5U / L, which was higher than that of immature DC's CD40CD80C (9.7 鹵0.5 鹵7.5 鹵1.2 鹵1.2 鹵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 鹵0.5 鹵7.5 鹵1.2 鹵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.
【作者單位】： 廣州軍區(qū)廣州總醫(yī)院皮膚科;
【基金】：國家自然科學(xué)基金(81071401)
【分類號】：R392
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本文編號：1503495