Molecular biological and biochemical study of plasma Platelet-activating factor-acetylhydrolase ; its regulation and function

1.血浆血小板活化因子-乙酰水解酶cDNA的分子克隆纯化的豚鼠血浆血小板活化因子-乙酰水解酶（Mw 63 kDa）用赖氨酸内肽酶酶切。用反相高效液相色谱法分离得到9个肽段，用气相氨基酸测序仪测定氨基酸序列。根据肽段序列合成得到的寡核苷酸引物，以豚鼠肝脏poly (A)^+ RNA第一链cDNA为模板进行PCR。扩增出约600 bp的特异片段。以PCR片段为探针，从30万pfu的豚鼠肝脏cDNA文库中筛选出4个阳性克隆。这些都不包含蛋白的n端区域，因此进行了5' RACE。该cDNA编码436个氨基酸（预计分子量为49 kDa），包含一个丝氨酸酯酶活性位点和三个假定的n链糖基化位点的一致序列。基因表达和抗体制备将全长cDNA连接在带有His标记的大肠杆菌表达载体上。重组蛋白具有血小板活化因子乙酰水解酶活性，通过Ni^+柱和SDS-PAGE纯化。用纯化后的蛋白免疫家兔，获得单特异性抗体。该抗体可识别豚鼠血浆中含有的63 kda蛋白，表明该酶经过糖基化修饰。这种翻译后修饰的意义还有待阐明。进行基因组DNA的克隆。最近，有报道称该酶的表达受肝脏和巨噬细胞中的雌二醇以及肥大细胞中的白细胞介素的调节。计划使用启动子区分析和免疫化学方法在分子水平上分析该酶的调控和功能。

1.Molecular cloning of the cDNA of plasma platelet-activating factor-acetylhydrolase Purified guinea pig plasma platelet-activating factor-acetylhydrolase (Mw 63 kDa) was digested with lysylendopeptidase. The 9 resulting peptides were isolated by reverse-phase HPLC,and their amino acid sequences were determined using a gas-phase amino acid sequencer. The deduced oligonucleotide primers were then syntesized on the basis of the petide sequences, and PCR was performed using first-strand cDNA derived from guinea pig liver poly (A)^+ RNA as a template. A specific fragment of about 600 bp was amplified. Four positive clones were screened from 300,000 pfu of a guinea pig liver cDNA library using the PCR fragment as a probe. None of these contained the N-terminal region of the protein, and 5' RACE was performed. The cDNA encoded 436 amino acids (predicted Mw 49 kDa) and contained consensus sequences for an active site of serine-esterases and three putative N-linked glycosylation sites.2.Gene expression and antibody preparationThe full-length cDNA was ligated in an E.coli expression vector with a His tag. The recombinant protein showed platelet-activating factor-acetylhydrolase activity, and was purified using a Ni^+ column and SDS-PAGE.The purified protein was used to immunize a rabbit, and a mono-specific antibody was obtained. This antibody recognized a 63-kDa protain contained in guinea pig plasma, suggesting that the enzyme was modified by blycosylation. The significance of this post-translational modification remaines to be elucidated.Cloning of the genomic DNA is performed. Recently, the expression of this enzyme was reported to be regulated by estradiol in the liver and macrophages and by interleukins in mast cells. Analysis of the regulation and function of this enzyme at the molecular level is planned using promoter region analysis and immunochemical methods.