CAJ | 학술논문

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 gD基因修饰的树突状细胞（ dendritic cell ， DC）疫苗的可行性。方法将HSV-2 gD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pAdeno-HSV-2 gD。分离小鼠骨髓DC细胞， pAdeno-HSV-2 gD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 gD的表达情况。结果以HSV-2病毒DNA为模板，采用gD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gD基因同预期的大小一致（1182 bp）。 pAdeno-gD DNA用脂质体法转染293细胞10 d，反复冻融获得pAdeno-HSV-2 gD重组腺病毒，活性为4×1010 IU／mL。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）。 pAdeno-HSV-2 gD-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 gD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pAdeno-HSV-2 gD-DC疫苗。
목적：생식기포진상무철저치유적방법。연제유효적생식기포진병독역묘시예방화치료HSV-2감염적관건，문중탐토제비선병독개도적HSV-2 gD기인수식적수돌상세포（ dendritic cell ， DC）역묘적가행성。방법장HSV-2 gD단백기인극륭도질립재체Shuttle-2，중조질립매절감정、측서감정후구건중조선병독pAdeno-HSV-2 gD。분리소서골수DC세포， pAdeno-HSV-2 gD전염DC세포후용류식세포술검측DC표형，용면역조화법、RT-PCR、SDS-PAGE화Western blot 방법검측HSV-2 gD적표체정황。결과이HSV-2병독DNA위모판，채용gD기인인물확증출상응적목적편단。경지당응효전영현시，PCR산물gD기인동예기적대소일치（1182 bp）。 pAdeno-gD DNA용지질체법전염293세포10 d，반복동융획득pAdeno-HSV-2 gD중조선병독，활성위4×1010 IU／mL。류식세포의검측결과현시：성숙DC화선병독감염후DC，CD40함량위（74．2±3．9）％、（81．3±3．1）％，CD80함량위（73．9±4．1）％、（80．4±2．9）％，CD86함량위（76．1±5．5）％、（83．7±3．9）％，균교불성숙DC적CD40、CD80、CD86함량[（9．7±0．5）％、（7．5±1．2）％、（5．2±1．1）％]승고（P＜0．01）。 pAdeno-HSV-2 gD-DC화세포인자자격유도성숙적DC세포표면분자표체차이무통계학의의（P＞0．05）。 RT-PCR、면역조직화학방법증명HSV-2 gD능재DC세포내표체，표체산물적SDS-PAGE화Western blot분석발현，재상대분자질량위43000처유외원단백표체，여예기단백조대일치。결론성공제비료pAdeno-HSV-2 gD-DC역묘。
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] %) (P<0.01).No statistically significant differ-ences were found between the expression of the surface molecules in the pAdeno -HSV-2 gD DCs and that in the cytokine stimulation-in-duced mature DCs (P>0.05).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.