Rab4蛋白参与DC细胞内源性抗原递呈  

Rab4 and endogenous antigen presentation in dendritic cells

作　　者：柴琳琳[1] 万瑛[1] 邹丽云[1] 张晋宇[1] 李娜[1] 刘婷[1] 李景怡[1] 吴玉章[1] 

机构地区：[1]第三军医大学基础部全军免疫学研究所,重庆400038

出　　处：《免疫学杂志》2007年第3期239-242,247,共5页Immunological Journal

基　　金：国家自然科学基金资助项目(30400392)

摘　　要：目的建立稳定表达卵白蛋白的DC细胞株DC-OVA,研究Rab蛋白对DC内源抗原递呈的影响。方法首先构建含OVA蛋白基因慢病毒表达质粒pLentimycOVA;以DNA-磷酸钙共沉淀法转染293FT细胞制备慢病毒,用病毒感染DC2.4细胞及杀稻瘟毒素(Blasticidin)筛选方法建立稳定表达OVA的细胞株,Western blot鉴定DC-OVA细胞中OVA蛋白表达;然后用识别MHC I类分子-OVA肽复合物的T细胞杂交瘤B3Z细胞以及针对该复合物的单抗25D1.16检测DC-OVA细胞表面MHC-OVA肽复合物的形成情况,建立内源性抗原呈递的检测方法;最后采用脂质体法转染化学合成的Rab4、Rab5A、Rab7、Rab11的siRNA于DC-OVA中,B3Z检测DC内源抗原递呈的变化。结果酶切和测序分析证实转移质粒克隆成功,Western blot可在DC-OVA细胞中检测到OVA蛋白,B3Z细胞和25D1.16单抗可检测到DC-OVA细胞表面存在MHC I类分子-OVA表位多肽复合物,表明内源性抗原呈递系统成功建立。在此基础上,发现下调DC细胞中Rab4蛋白的表达,DC-OVA细胞刺激B3Z生成IL-2(白细胞介素2)的量明显下降。结论成功构建了OVA蛋白的慢病毒表达载体,获得了表达内源OVA蛋白的DC细胞株,建立了内源性抗原呈递系统,初步证实下调Rab4蛋白可抑制DC细胞的内源性抗原递呈,为病毒感染和肿瘤等疾病的治疗奠定了基础。Objective To construct a dendritic cell llne DC-OVA for stable expression of ovalbumin （OVA）, and investigate the relationship between Rab and endogenous antigen presentation in dendritic cells. Methods ①A lentivirus expression vector pLentimycOVA was constructed. ②The three plasmids producing lentivirus （packaging plasmid, envelope plasmid, and target plasmid pLentimycOVA） were packaged into virus packaging cell line 293FT using DNA-Ca3 （PO4）2 co-transfection method to produce the lentivirus.③DC2.4 cells were infected by lentivirus and selected by Blasticidin. The OVA expression and endogenous presentation in DC-OVA cells were detected by B3Z, FACS, and Western blotting. ④The siRNAs against Rab4, Rab5A, Rab7, and Rabll were transfected into DC-OVA by liposome-mediated gene transfer method. After transfection, the endogenous antigen presentation of DC-OVA was measured with IL-2 level produced by B3Z. Results Recombinant was confirmed using restriction enzymes and DNA sequencing. OVA was detected in the lysates of DC-OVA. The IL-2 produced by DC-OVA-stimulated B3Z and the Rab4 expression in DC cells were declined. Conclusion The lentivirus expression plasmids pLentimycOVA is constructed successfully; the cell line endogenous expressing OVA and the detection system are established. The primary experiments suggest that the decrease of Rab4 can inhibit endogenous antigen presentation of DC, which provides a found for therapy of virus infection and tumors.

关 键 词：DC OVA 内源 抗原递呈 Rab4 

分 类 号：R392[医药卫生—免疫学]