SYNTHESIS AND EXPRESSION IN ESCHERICHIA-COLI OF THE GENE ENCODING MONOCYTE-DERIVED NEUTROPHIL-ACTIVATING FACTOR - BIOLOGICAL EQUIVALENCE BETWEEN NATURAL AND RECOMBINANT NEUTROPHIL-ACTIVATING FACTOR
SYNTHESIS AND EXPRESSION IN ESCHERICHIA-COLI OF THE GENE ENCODING MONOCYTE-DERIVED NEUTROPHIL-ACTIVATING FACTOR - BIOLOGICAL EQUIVALENCE BETWEEN NATURAL AND RECOMBINANT NEUTROPHIL-ACTIVATING FACTOR
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DOI：
10.1073/pnas.85.23.9199
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发表时间：
1988-12-01
影响因子：
11.1
通讯作者：
BAGGIOLINI, M
中科院分区：
文献类型：
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作者：
LINDLEY, I;ASCHAUER, H;BAGGIOLINI, M
从脂多糖刺激的人单核细胞条件培养液中纯化的中性粒细胞激活因子(NAF)进行了测序，发现由72个氨基酸组成：SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS.天然NaF的纯化制剂除了这种主要形式外，还含有少量的三个氨基末端变体，分别为77(+AVLPR)、70和69个残基。合成了编码72个氨基酸的NaF基因，并在大肠杆菌中进行了表达。用抗天然NaF的抗血清对细菌粗提物进行免疫印迹分析，发现有一条与天然NaF迁移的单一条带。纯化后的重组NaF的氨基和羧基末端序列与72个氨基酸的天然NaF完全相同。重组NaF在人中性粒细胞上的实验表明，重组NaF具有与天然NaF相同的活性和效力，可诱导细胞趋化，迅速增加胞内游离钙离子，激活呼吸爆发，释放特异性和亲天蓝颗粒内容物。
The neutrophil-activating factor (NAF) purified from the conditioned medium of lipopolysaccharide-stimulated human monocytes was sequenced and found to consist of 72 amino acids: SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS. Purified preparations of natural NAF contained, in addition to this main form, minor amounts of three amino-terminal variants with 77 (+AVLPR), 70, and 69 residues. A gene coding for the 72-amino acid NAF was synthesized, cloned and expressed in Escherichia coli. Western (immunologic) blot analysis of crude bacterial extracts, with an antiserum raised against natural NAF, revealed a single band that comigrated with natural NAF. Recombinant NAF purified to homogeneity had identical amino- and carboxyl-terminal sequences to the 72-amino acid natural NAF. Recombinant NAF was tested on human neutrophils and had the same activity and potency as natural NAF in inducing chemotaxis, rapidly increasing cytosolic free Ca2+, activating the respiratory burst, and releasing specific and azurophilic granular contents.