Melrose J, Smith S, Rodgers K, Little C, Burkhardt D, Ghosh P
The Institute of Bone and Joint Research, The Royal North Shore Hospital of Sydney, and Department of Surgery of The University of Sydney, St. Leonards, NSW, Australia.
Histochem Cell Biol. 2000 Aug;114(2):137-46. doi: 10.1007/s004180000173.
A polyclonal anti-bovine pancreatic trypsin inhibitor (BPTI) IgY was raised in chickens immunised with aprotinin. The anti-BPTI IgY was subsequently isolated from egg yolks and purified to homogeneity by affinity chromatography on immobilised aprotinin and by Superose 6 size exclusion fast protein liquid chromatography (FPLC). Immunoblotting with the chicken IgY demonstrated its specificity for BPTI; 3.9 ng BPTI could be detected by this technique. There was no crossreactivity against alpha1-proteinase inhibitor (human and sheep), inter-alpha-trypsin inhibitor (human and sheep), secretory leucocyte proteinase inhibitor or a range of serine proteinase inhibitory proteins (SPIs) isolated from plant sources (soybean and lima bean trypsin inhibitor, potato trypsin and chymotrypsin inhibitors) or serum SPIs (antithrombin-III, alpha2-macroglobulin). Immunoblotting using the anti-BPTI IgY identified the 6- to 12- and 58-kDa forms of endogenous ovine cartilage SPIs in cartilage extracts, confirming the interrelationship of the ovine cartilage SPIs with BPTI. BPTI-domain SPIs were immunolocalised within mast cells of ovine and bovine duodenum, lung and pancreas, and in ovine and bovine bronchial cartilage chondrocytes, chondrocytes of the superficial and intermediate zones of articular cartilage and in the fibrochondrocytes/chondrocytes of the nucleus
用抑肽酶免疫鸡后产生了一种多克隆抗牛胰蛋白酶抑制剂（BPTI）IgY。随后从蛋黄中分离出抗BPTI IgY，并通过固定化抑肽酶亲和层析和Superose 6尺寸排阻快速蛋白质液相色谱（FPLC）将其纯化至同质。用鸡IgY进行免疫印迹证明了其对BPTI的特异性；用该技术可检测到3.9 ng BPTI。对α1-蛋白酶抑制剂（人和羊）、α-间胰蛋白酶抑制剂（人和羊）、分泌型白细胞蛋白酶抑制剂或从植物来源分离的一系列丝氨酸蛋白酶抑制蛋白（SPIs）（大豆和利马豆胰蛋白酶抑制剂、马铃薯胰蛋白酶和糜蛋白酶抑制剂）或血清SPIs（抗凝血酶III、α2-巨球蛋白）无交叉反应。使用抗BPTI IgY的免疫印迹在软骨提取物中鉴定出内源性绵羊软骨SPIs的6至12 kDa和58 kDa形式，证实了绵羊软骨SPIs与BPTI之间的相互关系。BPTI结构域的SPIs在绵羊和牛十二指肠、肺和胰腺的肥大细胞内，以及绵羊和牛支气管软骨软骨细胞、关节软骨浅表层和中间层的软骨细胞以及细胞核的纤维软骨细胞/软骨细胞中进行了免疫定位。
