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目的构建携带人白细胞介素-1受体拮抗剂(hIL-1Ra)或人白细胞介素-10(hIL-10)基因的重组逆转录病毒(rRV),体外转染兔滑膜成纤维样细胞,检测目的基因的表达水平与持续时间。方法提取人外周血单个核细胞总RNA,反转录聚合酶链反应(RT-PCR)扩增目的基因;酶切、连接目的基因与逆转录病毒载体pLXSN,筛出阳性克隆,经GP2-293细胞包装,收集病毒并鉴定;rRV-hIL-1Ra、rRV-hIL-10分别体外转染兔滑膜成纤维样细胞,RT-PCR测定目的基因mRNA表达水平与时间的关系。免疫组织化学与免疫印迹测定蛋白水平的表达与持续时间。结果成功构建了携带hIL-1Ra或hIL-10基因的重组逆转录病毒,rRV-hIL-1Ra与rRV-hIL-10均能有效转染体外培养的兔滑膜成纤维样细胞,RT-PCR测定目的基因mRNA高峰均出现于转染后第5天左右。免疫组织化学和免疫印迹均检测到hIL-1Ra、hIL-10的表达。经G418筛选后的细胞中,hIL-1Ra的表达在转染后第30天内达高峰,至少持续60d;hIL-10的表达至少持续40d。结论以重组逆转录病毒为载体可以成功地将hIL-1Ra或hIL-10基因导入体外培养的兔滑膜成纤维样细胞并实现稳定表达。
Objective To construct recombinant retrovirus (rRV) carrying human interleukin-1 receptor antagonist (hIL-1Ra) or human interleukin-10 (hIL-10) gene and transfect rabbit synovial fibroblast- , Detect the expression level and duration of the target gene. Methods The total RNA of human peripheral blood mononuclear cells (PBMC) was extracted and the target gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The target gene was cloned and ligated with the retrovirus vector pLXSN. The positive clones were screened by GP2-293 The cells were harvested and virus was collected and identified. Rabbit synovial fibroblast-like cells were transfected with rRV-hIL-1Ra and rRV-hIL-10 respectively. The expression of target gene mRNA was analyzed by RT-PCR. Immunohistochemistry and Western blotting were used to determine the protein expression and duration. Results Recombinant retrovirus carrying hIL-1Ra or hIL-10 gene was successfully constructed. Both rRV-hIL-1Ra and rRV-hIL-10 were successfully transfected into rabbit synovial fibroblast-like cells in vitro. RT- The peak of mRNA of the target gene appeared on the 5th day after transfection. The expressions of hIL-1Ra and hIL-10 were detected by immunohistochemistry and Western blot. After G418 screening, hIL-1Ra expression peaked at 30 days after transfection for at least 60 days; hIL-10 expression persisted for at least 40 days. Conclusion The hIL-1Ra or hIL-10 gene can be successfully transfected into rabbit synovial fibroblast-like cells with recombinant retroviral vector and stably expressed.