互补DNA
生物
分子生物学
重组DNA
转染
细胞培养
克隆(编程)
免疫印迹
分子克隆
自然杀伤细胞
基因
生物化学
体外
细胞毒性
遗传学
程序设计语言
计算机科学
作者
Stanley F. Wolf,Patricia A. Temple,Michiko Kobayashi,Deborah Young,M Dicig,Leslie Lowe,Robin C. Dzialo,Lori Fitz,Catherine R. Ferenz,Rodney M. Hewick
出处
期刊:Journal of Immunology
[The American Association of Immunologists]
日期:1991-05-01
卷期号:146 (9): 3074-3081
被引量:752
标识
DOI:10.4049/jimmunol.146.9.3074
摘要
Abstract Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.
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