摘要
克隆小鼠白细胞介素 18(IL 18)编码区的cDNA ,并实现在真核细胞中的表达。方法 :用小鼠白细胞介素 18(mIL 18)的cDNA核苷酸序列 ,设计、合成基因序列特异性引物 ,应用逆转录多聚酶链反应RT PCR ,以植物血凝 (PHA)和细菌脂多糖 (LPS)刺激的小鼠非粘附性脾细胞的mRNA为模板 ,扩增获得全长mIL 18,转染小鼠成纤维细胞系SVT2 ,并进行IL 18生物学活性的检测。结果 :经测序证实获得的小鼠IL 18的cDNA序列与文献报道的mIL 18的cDNA序列完全一致。构建的小鼠IL 18的重组表达载体在转染小鼠成纤维细胞SVT2后 ,获得具有生物学活性mIL 18的分泌表达。结论 :克隆了小鼠IL 18的cDNA ,并实现了在真核细胞中的表达。
Objective:To clone murine interleukin-18 cDNA and express it by transfection of murine fibroblast SVT2. Methods:According to the previously reported nucleic acid sequence of murine interleukin-18(mIL-18) cDNA, specific primers for mIL-18 have been designed and synthesized. Using total RNA from nonadherent splenocytes stimulated with PHA and LPS as the template, mIL-18 cDNA was amplified by reverse transcription polymerase chain reaction(RT-PCR). The mIL-18 eukaryotic expression vector was constructed by subcloning this cDNA fragment into pcDNA3 at EcoRI/XbaI restriction sites, and use the recombinat vector to transfect murine fibroblast cell line SVT2, secreted mIL-18 level was determined. Results:Sequence analysis indicated that this cDNA is 100% homology tothe published mIL-18 cDNA sepuence, and found biological mIL-18 expressed in the supernatants of the transfected cells.Conclusion: We have cloned murine IL-18 cDNA and exqressed it by murine fibroblast SVT2.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2000年第5期252-254,共3页
Chinese Journal of Immunology
