小鼠CCR7基因重组腺病毒的构建及其在DC2.4的表达  

Construction of recombinant adenovirus carrying murine CCR7 gene and its expression on DC2.4

作　　者：应林燕[1] 符州[1] 罗健[1] 陈艳[2] 王莉佳[3] 李欣[3] 

机构地区：[1]重庆医科大学附属儿童医院呼吸中心,400014 [2]上海复旦大学附属儿科医院,201102 [3]重庆医科大学附属儿童医院发育疾病研究省部共建教育部重点实验室,400014

出　　处：《免疫学杂志》2010年第11期925-929,共5页Immunological Journal

基　　金：国家自然科学基金(30600684)

摘　　要：目的构建含有小鼠趋化因子受体-7（CCR7）基因的重组腺病毒（AdCCR7）,观察其体外感染DC2.4细胞效率。方法将小鼠CCR7基因克隆到穿梭质粒pAdTrack-CMV,在BJ5l83菌内和骨架质粒pAdeasy-1同源重组,筛选阳性克隆;经线性化后转染HEK293细胞,获得含小鼠CCR7基因的重组腺病毒AdCCR7;TCID50方法检测病毒滴度,PCR法鉴定病毒DNA。带绿色荧光蛋白腺病毒（AdGFP）和AdCCR7分别感染DC2.4（GFP-DC2.4和CCR7-DC2.4）,同时设未处理DC2.4为空白对照。流式细胞术（FCM）检测各组细胞表面分子CD11c,MHCⅡ,CD86及CCR7表达,趋化实验检测CCR7的功能。结果获得滴度约为l.4×1010pfu/ml重组腺病毒AdCCR7。AdCCR7感染DC2.4后,CCR7-DC2.4组与另外两组细胞比较CD11c、MHCⅡ及CD86的表达均无明显差异,但CCR7表达升高;其对趋化因子CCL19的趋化率可达44.7%~60.0%,明显高于其它两组。结论成功构建含小鼠CCR7基因的重组腺病毒AdCCR7,该病毒可感染细胞株DC2.4,增加细胞表面CCR7表达以及细胞对CCL19的趋化性,为进一步体内实验研究携带CCR7基因的未成熟DCs功能提供了工作基础。In this study,we aimed to construct and identify the recombinant adenovirus carrying murine chemokine receptor-7（mCCR7） genes and express it on DC2.4.Mouse CCR7 gene amplified by reverse transcription PCR was subcloned to the shuttle plasmid pAdTrack-CMV.Then the linearized shuttle plasmid was homogenously recombined with backbone plasmid pAdeasy-1 in E.coli BJ5183,and the candidate clone was further analyzed by restriction endonuclease digestion.The recombined plasmid was transfected into HEK293 cells by Lipofectamine2000 for packaging and amplifying.Virus titer was detected using the method of TCID50 and virus was identified by extraction of DNA for PCR.GFP-DC2.4 and CCR7-DC2.4 cells were obtained by infecting DC2.4 cells with AdGFP and AdCCR7 separately,with untreated DC2.4 as control.The expressions of surface molecules CD11c,MHCⅡ,CD86,and CCR7 were detected by FCM;CCR7 function was detected by chemotaxis assay.Results showed that the mCCR7 gene was cloned into recombinant adenovirus successfully（AdCCR7） and viral titer was about l.4 × 1010 pfu/ml.CCR7-DC2.4 demonstrated no difference in the expression of CD11c,MHC Ⅱ and CD86,as compared with GFP-DC2.4 and DC2.4,but showed higher expression of CCR7.Chemoattractant rate of CCR7-DC2.4 to CCL19 was 44.7% to 60%,significantly higher than that of the other two groups.We conclude that the constructed recombinant adenovirus containing mCCR7 could infect DC2.4,increase the expression of CCR7 and the cell chemotaxis to CCL19,which may lay a foundation to investigate the function of imDC transfected by mCCR7 gene in vivo.

关 键 词：CCR7 DC2.4 腺病毒 

分 类 号：R392.12[医药卫生—免疫学]