摘要
目的构建小鼠白细胞介素18(interleukin18,IL-18)的真核表达质粒,并检测其表达的IL-18的生物学活性。方法采用RT-PCR获得小鼠IL-18基因,通过EcoRⅠ和HindⅢ双酶切及连接反应,构建pEGFP-mIL-18真核表达载体,重组载体经过限制性内切酶、PCR及DNA序列测定等证实连接片段的正确性后,转染HEK293细胞系,荧光显微镜观察GFP-mIL-18融合蛋白的表达,收集转染后72h的上清液,MTT法检测上清液中IL-18表达产物的生物学活性。结果酶切分析、PCR鉴定、DNA测序表明成功地构建了pEGFP-mIL-18真核表达载体,MTT法证明表达产物有刺激小鼠脾细胞增殖的功能。结论所获pEGFP-mIL-18真核表达载体能在体外表达具有生物学活性的IL-18,为进一步研究IL-18的功能和运用奠定了基础。
OBJECTIVE To construct a eukaryotic expression vector containing mouse interleukin 18（IL- 18）, and examine the bioactivity of the IL- 18 expressed. METHODS The cDNA of mlL- 18 was cloned through reverse transcription - polymerase chain reaction（ RT- PCR）. The pEGFP-mlL- 18 eukaryotic expression vector was constructed through EcoR I and HindⅢ double enzyme digestion and ligation, and the recombination was identified by enzyme digestion , PCR, and DNA sequencing. Cell supematants was collected 72 hours after the plasmid pEGFP- mlL- 18 was transformed into 293 cell line. The bioactivity of the expressed IL - 18 in the supernatants was examined by MTr method. RESULTS The plasmid pEGFP - mIL - 18 was transformed into 293 cell line successfully, the expression of GFP - mIL - 18 recom- bined protein was observed with immunofluorescence. The expressed protein was proved to be able to prompt the proliferation of murine splenocytes by MTT method. CONCLUSION The pEGFP - mIL- 18 eukaryotic expression vector was constructed correctly;the GFP- mIL- 18 recombined protein is expressed and it can prompt the proliferation of murine splenocytes.
出处
《华西药学杂志》
CAS
CSCD
北大核心
2005年第6期476-479,共4页
West China Journal of Pharmaceutical Sciences
