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目的:分离和鉴定人类IL-10、IL-10受体(IL-10Rα)、IL-4及其受体IL-4Rα启动子序列,并分析IL-10、IL-4以及其受体的启动子在人类单纯疱疹病毒1型(HSV-1)激活卡波济肉瘤相关疱疹病毒(KSHV)过程中的启动活性。方法:以人类基因组DNA为模板,PCR扩增IL-10、IL-10Rα、IL-4Rα启动子区序列,并分别克隆载入pGL-3基本载体中虫荧光素酶(Luciferase)报告基因的上游。进一步将上述构建的重组质粒与本室保存的含IL-4启动子重组质粒分别转染HSV-1感染的BCBL-1细胞,并作Luciferase活性检测。结果:分离了IL-10、IL-10Rα和IL-4Rα的启动子区域序列,并成功克隆了含启动子序列重组报告质粒;HSV-1感染的BCBL-1细胞在进一步转染了IL-10、IL-10Rα、IL-4和IL-4Rα启动子重组报告质粒后,其Luciferase值与相应的对照比较显著升高(P<0.05)。结论:在BCBL-1细胞中,HSV-1可通过直接激活IL-10、IL-4以及相应受体的启动子来上调它们的表达;在HSV-1感染的BCBL-1细胞中,IL-10和IL-4可能对KSHV的裂解复制起到促进作用。
OBJECTIVE: To isolate and identify human IL-10, IL-10 receptor (IL-10Rα), IL-4 and their receptor IL-4Rα promoter sequences and analyze the initiation of IL-10, IL-4 and their receptors Promoter activity in the activation of Kaposi’s sarcoma-associated herpesvirus (KSHV) by human herpes simplex virus type 1 (HSV-1). Methods: Human genomic DNA was used as a template to amplify the IL-10, IL-10Rα and IL-4Rα promoter regions and cloned into the upstream of the luciferase reporter gene in the pGL-3 basic vector . Further, the recombinant plasmid constructed above and the recombinant plasmid containing IL-4 promoter preserved in our laboratory were respectively transfected into HSV-1-infected BCBL-1 cells for Luciferase activity assay. Results: The promoter region sequences of IL-10, IL-10Rα and IL-4Rα were isolated and successfully cloned the recombinant plasmid containing the promoter sequence. The HSV-1-infected BCBL-1 cells were further transfected with IL-10 , Luciferase of IL-10Rα, IL-4 and IL-4Rα promoter were significantly increased compared with the corresponding control (P <0.05). CONCLUSIONS: In BCBL-1 cells, HSV-1 up-regulates the expression of IL-10, IL-4 and its corresponding receptor by activating the promoters of IL-10 and IL-4. In HSV-1 infected BCBL- 10 and IL-4 may promote the cleavage and replication of KSHV.