Fransen L, Müller R, Marmenout A, Tavernier J, Van der Heyden J, Kawashima E, Chollet A, Tizard R, Van Heuverswyn H, Van Vliet A
Nucleic Acids Res. 1985 Jun 25;13(12):4417-29. doi: 10.1093/nar/13.12.4417.
Tumour necrosis factor (TNF), released by induced macrophages, causes tumour necrosis in animals and kills preferentially transformed cells in vitro. mRNA induced in the established mouse monocytic PU 5.1.8 cell line by lipopolysaccharide, was converted into double-stranded cDNA and cloned in the pAT153 vector. Recombinant plasmids were screened by plus-minus hybridization and TNF-specific oligonucleotide probes constructed on the basis of partial amino acid sequences of rabbit TNF. A series of TNF specific clones were identified and confirmed by hybrid selection of mouse TNF-specific mRNA. The sequence codes for a 235 amino acids long polypeptide, of which 156 amino acids presumably correspond to the mature product. It can be concluded that mature mouse TNF is a glycosylated dimer. Biologically active TNF was secreted by both Cos-I and CHO-cells transfected with the chimaeric expression vector pSV2d2-mTNF containing the coding region of the mouse TNF cDNA gene.
由诱导的巨噬细胞释放的肿瘤坏死因子（TNF）可导致动物体内肿瘤坏死，并在体外优先杀死转化细胞。脂多糖诱导已建立的小鼠单核细胞PU 5.1.8细胞系产生的mRNA，被转化为双链cDNA并克隆到pAT153载体中。通过正负杂交筛选重组质粒，并根据兔TNF的部分氨基酸序列构建TNF特异性寡核苷酸探针。通过小鼠TNF特异性mRNA的杂交选择鉴定并确认了一系列TNF特异性克隆。该序列编码一个235个氨基酸长的多肽，其中156个氨基酸可能对应于成熟产物。可以得出结论，成熟的小鼠TNF是一种糖基化二聚体。用含有小鼠TNF cDNA基因编码区的嵌合表达载体pSV2d2-mTNF转染的Cos-I细胞和CHO细胞均分泌具有生物活性的TNF。
