人白细胞介素-1受体拮抗剂或白细胞介素-10逆转录病毒载体的构建与体外实验

Construction of two retroviral vectors for interleukin-1 receptor antagonist and interleukin-10 and in vitro transfection of rabbit synoviocytes

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。

Objectives To construct two retroviral vectors, one containing human interleukin-1 recep-tor antagonist (hIL-1Ra) gene and the other containing human interleukin-10 (hIL-10) gene and to transfect rabbit synoviocytes in vitro and detect the expression level of target genes. Methods RNA from human peripheral blood mononuclear cells were extracted and target genes were amplified by RT-PCR. The target genes were cloned into retroviral vector pLXSN, which was then transducted into GP2-293 cells to produce recombinant retrovirus. Rabbit synoviocytes were transfected and the expression of target genes was detected by RT-PCR, immunohistochemistry and western-blot. Results The retroviral vector containing hIL-1Ra gene or hIL-10 gene was constructed successfully. The hIL-1Ra gene and hIL-10 gene were transduced respectively into rabbit synoviocytes in vitro. The mRNA level of both genes reached peak in 5 days. In positive cell clones, the protein level of hIL-1Ra reached peak within 30 days and maintained at least 60 days; the protein level of hIL-10 maintained at least 40 days. Conclusion The hIL-1Ra gene and hIL-10 gene can be transduced successfully into rabbit synoviocytes by recombinant retrovirus.