摘要
目的构建大鼠趋化因子CCL19表达慢病毒，建立CCL19体外表达模型。方法以CCL19亚克隆载体为模板，聚合酶链反应（PCR）扩增和双酶切连接构建CCL19慢病毒包装载体，采用第3代慢病毒包装系统制备CCL19慢病毒颗粒，定量PCR方法检测病毒滴度，Westernblot法检测CCL19在IEC6细胞中的表达。分离大鼠树突状细胞，并采用侵袭小室（Transwell）实验检测慢病毒感染的IEC6细胞对树突状细胞趋化功能的影响。结果PCR和测序结果表明326bp的CCL19基因表达序列连入慢病毒表达载体，质粒构建正确，定量PCR检测结果表明病毒包装滴度达到2×10^9TU／ml。荧光显微镜观察表明慢病毒体外感染IEC6获得80％以上的阳性表达率。Westernblot检测验证了CCL19蛋白在表达慢病毒感染的IEC6细胞中高表达。Transwell检测表明过表达CCL19的IEC6细胞可以提高树突状细胞的净迁移率达4倍以上。结论成功制备高滴度的CCL19表达慢病毒，并在IEC6细胞中检测到CCL19蛋白高表达，体外条件下CCL19的IEC6细胞可以提高树突状细胞趋化性。
Objective To construct CCL19 expression lentivirus and establish in vitro model of CCL19 expression. Methods The CCL19 gene cDNA was amplified from subclone vector, double cut and linked to construct lentiviral vector. The third generation envelope system was used to produce lentiviral particles. Titers of virus were determined by quantitative polymerase chain reaction （PCR） and protein expression of CCL19 in IEC6 cells was detected by using Western blotting. Rat dendritic cells were isolated and Transwell was used in lentivirus infected IEC6 cells to evaluate chemotaxis. Results PCR and sequencing confirmed that 326 bp cDNA of CCL19 was linked into vector and the rat chemokine CCL19 lentivirual vector was successfully constructed. Titer of lentivirus was determined at 2×10^9TU/ml via quantitative PCR. Fluorescent observation showed that 80% expression rate was obtained in IEC6 infection in vitro. Results also demonstrated that Western blotting confirmed CCL19 protein expression in infected rat intestinal epithelial cells. Traswell presented notable increase of dendritic cells net immigration by 4 fold when CCL19 was overexpressed in IEC6 cells. Conclusion CCL19 expression lentivirus was successfully constructed and CCL19 protein over-expression was detected. The chemotaxis of dendritic cells was stimulated by CCL19 overexpression in IEC6 ceils.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第4期636-639,共4页
Chinese Journal of Experimental Surgery
基金
福建省科技计划重点资助项目（2008Y0043）
