趋化因子受体7基因绿色荧光慢病毒载体构建及在未成熟树突状细胞中的表达**

背景：趋化因子受体7(chemokine receptor-7, CCR7)是树突状细胞从外周迁移至淋巴系统发挥作用的最重要的启动和调节者,但未成熟树突状细胞表面不表达CCR7,因此利用携带CCR7基因的未成熟树突状细胞可以更好地诱导免疫耐受。目的：构建携带小鼠CCR7基因的绿色荧光蛋白重组慢病毒载体,观察其在未成熟树突状细胞中的表达。方法：采用RT-PCR扩增小鼠CCR7基因并克隆至pCR-Blunt载体。将CCR7 DNA片段及IRES-GFP连入慢病毒转移质粒LV-Lac,生成重组慢病毒质粒LV-CCR7。采用脂质体转染法将慢病毒系统3质粒(重组慢病毒质粒LV-CCR7、包装质粒     ΔNRF及包膜质粒pVSVG)共转染包装慢病毒,重组慢病毒感染未成熟树突状细胞,光学显微镜观察细胞状态,流式细胞术鉴定CCR7蛋白的表达。结果与结论：实验成功扩增出小鼠CCR7 DNA片段并克隆至pCR-Blunt载体,亚克隆构建慢病毒表达载体LV-CCR7,经3质粒包装系统感染293 FT细胞后,24 h于荧光显微镜下均观察到绿色荧光蛋白阳性表达,病毒滴度为108 U/L以上,获得携带CCR7基因的重组慢病毒。慢病毒颗粒可有效感染未成熟树突状细胞,荧光显微镜可见大量GFP蛋白表达,阳性细胞达50%,流式细胞术检测到CCR7蛋白表达,LV-CCR7基因修饰的未成熟树突状细胞仍保持在未成熟状态。结果证实,实验成功构建携带小鼠CCR7基因绿色荧光慢病毒载体LV-CCR7,并可在未成熟树突状细胞细胞中表达。关键词：趋化因子受体7;未成熟树突状细胞;慢病毒载体;真核表达;组织工程doi:10.3969/j.issn.1673-8225.2012.01.029

Construction and expression of mouse chemokine receptor-7 green fluorescent lentiviral expression vector in immature dendritic cells

BACKGROUND: Chemokine receptor 7 (CCR7) plays a key role in launching and regulating the migration of dendritic cells (DCs) from peripheral tissueto the lymphatic system. But immature dendritic cells (imDCs)do not express CCR7. Therefore, imDCs carrying CCR7 possess great prospect for stronger immune tolerance.   OBJECTIVE: To construct green fluorescent protein (GFP) lentiviral expression vector with mouse CCR7 gene and to observe the expression of CCR7 gene in imDCs.METHODS: The CCR7 was amplified from the mouse thymus by RT-PCR and transfected into pCR-Blunt carrier. The CCR7 DNA fragment and IRES-GFP were cloned into lentiviral expression vector LV-Lac, to form recombinant lentivial plasmid LV-CCR7. Three plasmids of the lentivial system (recombinant lentivial plasmid LV-CCR7, package plasmid ΔNRF, envelope plasmid pVSVG) were co-transfected with package lentivirus by lipofectamine. The imDCs were infected by the recombinant lentivial, The morphology of transfected imDCs was examined under a fluorescent microscope and flow cytometry was used to determine the expression of CCR7.RESULTS AND CONCLUSION: The CCR7 fragment was amplified from cDNA of the mouse thymus and subcloned to pCR-Blunt carrier, and lentiviral expression vector LV-CCR7 was constructed successfully. After infected with 293 FT cells for 24 hours through three plasmid package system, positive expression of GFP was observed under fluorescence microscope. The titer of the lentivirus was above 108 U/L, and to obtain the recombinant lentivial with mouse CCR7 gene. The lentivial plasmid could infect imDCs effectively, a large amount of GFP and CCR7 expression was observed under fluorescence microscope and flow cytometry, respectively, and the positive rate was 50%. It indicates that GFP lentiviral vector LV-CCR7 with mouse CCR7 gene were successfully constructed and expressed in imDCs.