腺病毒介导HSV-2 gD基因修饰的树突状细胞疫苗制备  

Preparation of a dendritic cell vaccine modified by the adenovirus-mediated HSV-2 gD gene

作　　者：樊建勇[1] 王颖[1] 杨慧兰[1] 梁洁[1] 李翠华[1] 

机构地区：[1]广州军区广州总医院皮肤科

出　　处：《医学研究生学报》2015年第1期20-24,共5页Journal of Medical Postgraduates

基　　金：国家自然科学基金(81071401)

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(P<0.01)。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(P>0.05)。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。Objective Up to now, there has been no sure cure for genital herpes （GH）, and vaccine seems a most promis-ing approach to the prevention and treatment of herpes simplex virus Ⅱ（HSV-2） infection.In this study, we investigated the feasibili-ty of preparing a dendritic cell （ DC） vaccine modified by the adenovirus-mediated HSV-2 gD gene. Methods We subcloned the HSV-2 gD gene into the vector Shuttle-2 and constructed the recombinant adenovirus pAdeno-HSV-2 gD following identification by en-zyme digestion and DNA sequence analysis .We isolated DCs from the mouse bone marrow , analyzed their phenotypes by flow cytome-try after transfection with the recombinant adenovirus pAdeno-HSV-2 gD, and determined the expression of HSV-2 gD by immunohisto-chemistry, RT-PCR, SDS-PAGE, and Western blot. Results Based on HSV-2 DNA, the corresponding target fragments were am-plified with the gD gene primers.Agarose gel electrophoresis showed the correct size of the PCR product （1182 bp） as predicted.The recombinant adenovirus pAdeno-HSV-2 gD was obtained by transfecting the 293 cells with pAdeno-gD DNA, which had an activity of 4 ×1010 IU/mL.The contents of CD40, CD80, and CD86 were （74.2 ±3.9）, （73.9 ±4.1）, and （76.1 ±5.5） % in the mature DCs and （81.3 ±3.1）, （80.4 ±2.9）, and （83.7 ±3.9） % in the pAdeno-HSV-2 gD DCs, significantly increased as compared with those in the immature DCs （[9.7 ±0.5], [7.5 ±1.2], and [5.2 ±1.1] %） （P0.05）.RT-PCR and immunohistochemistry confirmed the expression of HSV-2 gD in DCs.SDS-PAGE and Western blot of the expressed protein showed a new band with an apparent molecular mass corresponding to the predicted size （43000）. Conclusion The results of our study have paved the ground for the successful preparation and identification of a dendritic cell vaccine modified by the adenovirus-mediated HSV-2 gD gene.

关 键 词：单纯疱疹病毒 树突状细胞 疫苗 

分 类 号：R373[医药卫生—病原生物学]