摘要
本文用重组iNOS基因真核表达载体转染NG108-15神经母细胞瘤和神经胶质瘤杂交细胞株,获得G418抗性克隆。在稳定表达iNOS基因2~#克隆中,胞浆相酶活性增加,伴有NO_2^-含量和胞内cGMP水平增高,提示iNOS基因表达参与NO-cGMP信号转导通路,且可被L-NNA和MB所阻断。蛋白表达产物的亚细胞定位分析显示功能性iNOS主要位于细胞胞浆中。对转染细胞做外源基因整合、转录和翻译水平鉴定,证实均有较高水平iNOS mR-NA转录和特异性蛋白表达,成功地建立了稳定表达iNOS基因的工程细胞。
The neuroblastoma X glioma NG108-15 cells were transfected with recombinant eukarytic expression plasmid pCMViNOS containing the full-length cDNA encoding inducible nitric oxide synthase (iNOS). A lot of G418-resistant clones were screened at 600μg/ml of geneticin. In the 2 clone expressing iNOS gene, iNOS catalytic activity in the cytosol fraction displayed to have an increasing trend, accompanying with the accumulation of NO2- content in the supernatant of cultured cells and the intracellular cGMP concentration, which suggested that NO-cGMP signal pathway was mediated by the expression of iNOS gene and blocked by Nc-nitro-L-arginine (L-NNA) and methylene blue (MB). Activity of iNOS was concentration-dependently inhibited by NOS inhibitors such as L-NNA and aminoguani-dine. The result of measurement of NADPH di-
aphorase activity and immunocytochemical staining showed that localization of the function expression of iNOS protein mainly existed in the cytoplasm of NG108-15 cells transfected with pCMViNOS. Furthermore, the chromosomal integration, transcript and protein translation of foreign iNOS gene were identified by Southern hybridization, RT-PCR and Western blot, respectively. The results indicated that iNOS gene-transfected cells had mRNA transcription and specific protein expression at high level. Given the above results, the engineering cell line with stable expression of iNOS gene was successfully established. The new neuronal cell line may serve as a source of iNOS and provide a useful cell model for studying iNOS biological function and developing novel iNOS-selective inhibitors.
出处
《实验生物学报》
CSCD
1999年第4期335-347,共13页
Acta Biologiae Experimentalis Sinica
基金
国家自然科学基金 No.39670827~~
关键词
INOS
NG108-15
基因表达
基因转移
抑制剂
Nitric-oxide synthase. Gene expression. Gene transfer. Signal transduction. Cyclic GMP. Neuroblastoma. Cell line.
