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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。
Objective To construct a recombinant adenovirus (AdCCR7) containing the chemokine receptor 7 (CCR7) gene and observe the efficiency of in vitro infection of DC2.4 cells. Methods The mouse CCR7 gene was cloned into the shuttle plasmid pAdTrack-CMV and homologously recombined with the plasmid pAdeasy-1 in BJ5183 to screen positive clones. After linearization, the recombinant plasmid was transfected into HEK293 cells to obtain recombinant adenovirus containing CCR7 gene Virus AdCCR7; TCID50 method for detecting virus titer, PCR method for the identification of viral DNA. DC2.4 (GFP-DC2.4 and CCR7-DC2.4) were infected with green fluorescent protein adenovirus (AdGFP) and AdCCR7 respectively, and untreated DC2.4 was also used as a blank control. The expression of CD11c, MHCⅡ, CD86 and CCR7 were detected by flow cytometry (FCM). The chemotaxis assay was used to detect the function of CCR7. As a result, a titer of about 1.4 × 10 <10> pfu / ml of recombinant adenovirus AdCCR7 was obtained. After AdCCR7 infection with DC2.4, the expression of CD11c, MHCⅡand CD86 were not significantly different between the CCR7-DC2.4 group and the other two groups, but the expression of CCR7 was increased. The chemotactic rate of chemokine CCL19 was 44.7 % ~ 60.0%, significantly higher than the other two groups. Conclusion Recombinant adenovirus AdCCR7 containing mouse CCR7 gene was successfully constructed and could infect DC2.4 cell line to increase the expression of CCR7 on cell surface and the chemotaxis of CCL19 cells. To further investigate the effect of CCR7 gene on immature DCs Function provides a working basis.