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目的:克隆小鼠次级淋巴样组织趋化因子(secondary lymph-oid-tissue chemokine,SLC)基因,并构建真核表达载体。在体 内外检测该表达载体表达产物的免疫趋化功能。方法:用RT-PCR从C57BL/6小鼠胸腺组织中克隆小鼠SLC基因,构建真核表达载体pcDNA3.1-mSLC。在体外,用基因枪转染小鼠黑色素瘤B16F10细胞;RT-PCR检测转染细胞有SLC的表达;利用趋化小室法,检测表达产物针对淋巴细胞的趋化活性。在体内,用基因枪通过小鼠皮肤局部转染SLC基因,观察转染局部淋巴细胞的浸润。结果:克隆的基因经测序证实,为小鼠SLC基因Scya21b型。转染SLC基因的B16F10细胞体外培养48 h后,RT-PCR检测到SLC的表达,同时培养基上清具有针对淋巴细胞的趋化活性。通过基因枪局部转染小鼠皮肤,24 h后皮肤病理检查显示,局部皮内有明显的淋巴细胞浸润。结论:从小鼠胸腺组织中克隆到小鼠SLC基因,构建的真核表达载体pcDNA3.1-mSLC在体内外均可以表达,并具有针对淋巴细胞的趋化活性。
Objective: To clone mouse secondary lymphoid tissue chemokine (SLC) gene and construct eukaryotic expression vector. The immune chemotactic function of the expression vector was tested in vivo and in vitro. Methods: Mouse SLC gene was cloned from C57BL / 6 mouse thymus by RT-PCR to construct eukaryotic expression vector pcDNA3.1-mSLC. In vitro, the murine melanoma B16F10 cells were transfected with gene gun; the expression of SLC was detected by RT-PCR; the chemotactic activity of the expressed products against lymphocytes was detected by chemotactic chamber method. In vivo, SLC genes were transfected locally into mouse skin using a gene gun to observe the infiltration of local lymphocytes. Results: The cloned gene was confirmed by sequencing and was Scya21b type of mouse SLC gene. The SLC gene was transfected into B16F10 cells for 48 h in vitro. The expression of SLC was detected by RT-PCR, and the supernatant of the medium had chemotactic activity against lymphocytes. By gene gun local transfection mouse skin, skin pathology examination after 24 h showed that the local intradermal lymphocyte infiltration. CONCLUSION: The mouse SLC gene was cloned from mouse thymus tissue and the eukaryotic expression vector pcDNA3.1-mSLC can be expressed both in vitro and in vivo with chemotactic activity against lymphocytes.