cDNA and genomic cloning and expression of the P48 monocytic differentiation/activation factor, a Mycoplasma fermentans gene product.
cDNA and genomic cloning and expression of the P48 monocytic differentiation/activation factor, a Mycoplasma fermentans gene product.
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P48 单核细胞分化/激活因子（一种发酵支原体基因产物）的 cDNA 和基因组克隆及表达。
DOI：
10.1042/bj3190919
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Chang,NS
中科院分区：
文献类型：
--
作者：
Hall,RE;Agarwal,S;Kestler,DP;Cobb,JA;Goldstein,KM;Chang,NS
P48 是一种 48 kDa 单核细胞分化/激活因子，之前从 Reh 人前 B 细胞白血病细胞系的条件培养基中纯化。它诱导 HL-60 人早幼粒细胞白血病细胞沿单核细胞途径生长停滞和分化，并在人单核细胞和单核细胞系中产生细胞因子白细胞介素 1、肿瘤坏死因子-α 和白细胞介素 6。 P48 的 cDNA 通过 cDNA 末端的 3´ 反向扩增从 Reh 细胞 RNA 中克隆。用 P48 cDNA 进行 Southern 印迹探测显示与 Reh 和 Molt-4 细胞的 DNA 杂交，但不与人外周血单核细胞的 DNA 杂交。随后使用 PCR 和 Southern 分析的研究揭示了从发酵支原体（而非发酵支原体）分离的 DNA 中存在 P48 序列。人型、爱荷华州型、滑液型型或 M.型。尽管使用支原体培养和杂交技术的初步研究未能揭示我们的 Reh 和 Molt-4 细胞系中的支原体感染，但随后使用支原体特异性 rRNA 引物的 PCR 研究揭示了这些细胞系中的支原体序列。使用 P48 cDNA 探针，我们从 M 中分离出一个基因组克隆。发现与 P48 cDNA 克隆有 98.5% 的同一性，推导的氨基酸序列与从 Reh 细胞系条件培养基中纯化的 P48 蛋白的 N 端微测序数据一致。该基因的5´端具有许多原核基因特有的共有序列，并且推导的氨基酸序列具有许多特征表明P48是脂蛋白。 P48 cDNA 在大肠杆菌的 pMAL 中表达，Western blot 发现表达的 60 kDa 融合蛋白与抗 P48 抗体发生反应。这与代表 42 kDa 麦芽糖结合蛋白和 18 kDa P48 重组蛋白总和的 pMAL 融合蛋白一致，表明天然 P48 具有显着的翻译后修饰。与此一致的是，Northern 印迹研究揭示了一个 1 kb 的转录本。发现重组融合蛋白具有针对 HL-60 细胞的抗增殖活性，并且发现针对重组 P48 的抗体可阻断从条件培养基中分离的天然 P48 的生物活性。这些研究表明，P48是一种具有免疫调节和造血分化活性的分子，来源于M.发酵罐或密切相关的物种。 P48 在支原体感染的病理生理学中可能很重要，并且可能有助于剖析涉及哺乳动物造血细胞分化、免疫功能和细胞因子生物合成的机制。
P48 is a 48 kDa monocytic differentiation/activation factor previously purified from the conditioned medium of the Reh human pre-B cell leukaemia cell line. It induces growth arrest and differentiation of HL-60 human promyelocytic leukaemia cells along the monocytic pathway and the production of the cytokines interleukin 1, tumour necrosis factor-α and interleukin 6 in human monocytes and monocytic cell lines. The cDNA for P48 was cloned from Reh cellular RNA using 3´ reverse amplification of cDNA ends. Southern blot probing with P48 cDNA revealed hybridization with DNA from Reh and Molt-4 cells, but not with DNA from human peripheral blood mononuclear cells. Subsequent studies using PCR and Southern analysis revealed P48 sequences in DNA isolated fromMycoplasma fermentansbut notM. hominis, M. iowae, M. synoviaeorM. lypophilum.Although initial studies usingMycoplasmaculture and hybridization techniques had failed to revealMycoplasmainfection in our Reh and Molt-4 cell lines, subsequent PCR studies usingMycoplasmagenus-specific rRNA primers revealedMycoplasmasequences in these cell lines. Using the P48 cDNA probe, we isolated a genomic clone fromM. fermentansDNA which was found to be 98.5% identical with the P48 cDNA clone, and the deduced amino acid sequence agreed with N-terminal microsequencing data for P48 protein purified from the Reh cell line conditioned medium. The 5´ end of the gene has a number of consensus sequences characteristic of prokaryotic genes, and the deduced amino acid sequence has a number of features suggesting that P48 is a lipoprotein. The P48 cDNA was expressed in pMAL inEscherichia coli, and the 60 kDa expressed fusion protein was found to react with anti-P48 antibodies on Western blots. This is consistent with a pMAL fusion protein representing the sum of the 42 kDa maltose-binding protein and 18 kDa of P48 recombinant protein, suggesting that native P48 has significant post-translational modification. Consistent with this, Northern blot studies revealed a single 1 kb transcript. The recombinant fusion protein was found to possess anti-proliferative activity against HL-60 cells, and antibodies against recombinant P48 were found to block the biological activity of native P48 isolated from conditioned medium. These studies demonstrate that P48, a molecule with immunomodulatory and haematopoietic differentiation activities, is derived fromM. fermentansor a closely related species. P48 may be important in the pathophysiology ofMycoplasmainfections and may be useful in dissecting the mechanisms involved in mammalian haematopoietic cell differentiation, immune function and cytokine biosynthesis.