摘要
白细胞介素 12 (IL - 12 )具有广谱抗肿瘤、抗感染的作用 ,是由两个亚单位 40kD(p40 )和 35kD(p35 )通过二硫键构成的杂合体 ,有可能通过分别表达亚单位的方式来表达有功能活性的IL - 12。该实验尝试利用重组腺伴随病毒 (rAAV)载体进行人IL - 12 (hIL - 12 )双亚基共表达 ,将hIL - 12的两个亚基分别克隆到AAV载体质粒pSNAV中 ,构建成 pSNAV -IL12 - p35和pSNAV -IL12 - p40质粒 ,经转染、G418筛选后建立了rAAV载体细胞株。采用先前建立的rAAV的生产方法 ,获得了rAAV -IL12 -p35和rAAV -IL12 - p40 ,在体外将两种rAAV共同感染BHK - 2 1细胞 ,48h后收集细胞培养上清进行免疫学和生物学活性检测。经ELISA检测 ,产生的hIL - 12 p70的含量为 10 185 pg/ml;在体外促进IFN -γ分泌实验中 ,加入hIL - 12的PBMC分泌的IFN -γ含量为 37 2mg/ml。实验结果说明 :采用两个AAV载体分别表达亚单位的方法可以表达具有功能活性的hIL - 12 ,为IL -
Interleukin-12(IL-12)is a kind of heterodimeric cytokine comprising p35 and p40 chains and has been demonstrated to be one of the most efficient antitumor cytokines in experimental animal models.It is possible to express the functional hIL-12 by expressing the two subunits separately.In our exeriment,we constructed the recombinant AAV(rAAV)vectors expressing either the IL 12 p35 or IL 12 p40 subunit. Infecting BHK-21 cells with rAAV-IL12-p35 and rAAV-IL12-p40,the media after 48 hours was collected for ELISA and biological tests and the positive results were obtained.The hIL-12 p70 produced was 10.185pg/ml and the hIL-12 stimulated PBMC could secrete IFN-γ about 37.2 mg/ml.These results suggest that biologically active hIL-12 can be produced by using two rAAV vectors to express two subunits,and it provides a new way to hIL-12 gene therapy
出处
《病毒学报》
CAS
CSCD
北大核心
2002年第1期52-56,共5页
Chinese Journal of Virology
