PREPARATION OF ANTIBODY PEPTIDE INHIBITING POLYPHENOL OXIDASE

我们计划利用从单抗中制备的抑制肽作为食品变质酶的抑制剂。多酚氧化酶(EC1.14.18.1)广泛存在于动植物中，催化食品中的褐变反应，是食品降解酶。1)由于从Sigma购买的酶制剂(蘑菇)是棕色的，含有污染蛋白，经DEAE-Sepharose和TSK-Gel-DEAE5PW离子交换层析和ZORBAX GF-1250.2凝胶过滤纯化多酚氧化酶，得到8个稳定的杂交瘤细胞株，在10个不同的融合株中获得了分泌抗纯化多酚氧化酶单抗的杂交瘤细胞株。这些抗体对酶的活性有抑制作用或刺激作用，或无影响。然而，其抑制活性很弱。因此，我们对免疫时间、免疫次数和佐剂种类进行了研究，但未能成功获得对该酶有强烈抑制作用的抗体。3)尝试从大肠杆菌中直接制备最小抗原结合单位Fv片段。从该酶免疫的小鼠脾中纯化出Poly(A)^+RNA，并逆转为cDNA.用合适的引物和聚合酶链式反应(PCR)成功地扩增出Fv基因。来自大肠杆菌的FV片段的表达正在进行中。

We planned to make use of an inhibitory peptide prepared from a monoclonal antibody as an inhibitor of food-deteriorating enzyme. Polyphenol oxidases (EC 1.14.18.1),which catalyze browning reaction in foods and are widely distributed throughout plants and mammals, were employed as food-deteriorating enzyme. 1) Since the enzyme preparation (mushroom) purchased from Sigma was brown-colored and contained the contaminating protein, polyphenol oxidase was purified to electrophoretic homogeneity by DEAE-Sepharose and TSKgel-DEAE 5PW ion exchange chromatography and gel filtration on ZORBAX GF-1250.2) Eight stable hybridoma cell lines which secrete monoclonal antibodies against purified polyphenol oxidase were obtained in ten separate fusions. These antibodies were inhibitory or stimulatory or had no effect on the enzyme activity. However, the inhibitory activity was very weak. Therefore, we investigated the term and the number of immunization and the kind of adjuvant, but we could not successfully obtain the antibody which strongly inhibits the enzyme. 3) We attempted to directly prepare Fv fragment, a minimum antigen-binding unit, from E. coli. Poly (A)^+RNA was purified from the spleen of the mouse immunized with the enzyme and were reversed to cDNA. Fv genes were successfully amplified using appropriate primers and polymerase chain reaction (PCR). Expression of the Fv fragment from E. coli is in progress.