PREPARATION OF ANTIBODY PEPTIDE INHIBITING POLYPHENOL OXIDASE

我们计划利用单克隆抗体制备的抑制肽作为食品变质酶的抑制剂。多酚氧化酶（EC 1.14.18.1）是广泛分布于植物和哺乳动物体内的一种催化食品褐变反应的变质酶。1)由于采购自Sigma的酶制品（蘑菇）为棕色且含有污染蛋白，采用DEAE-Sepharose和TSKgel-DEAE 5PW离子交换层析和ZORBAX gf - 12520凝胶过滤纯化多酚氧化酶，达到电泳均匀性。2)10次分离融合获得8株稳定的杂交瘤细胞系，可分泌抗纯化多酚氧化酶的单克隆抗体。这些抗体是抑制性或刺激性的，或对酶活性没有影响。但抑菌活性很弱。因此，我们研究了免疫期限、免疫次数和佐剂种类，但未能成功获得对该酶有强烈抑制作用的抗体。3)我们尝试直接从大肠杆菌中制备最小抗原结合单元Fv片段。Poly (A)^+RNA从经酶免疫的小鼠脾脏中纯化，并转化为cDNA。利用合适的引物和聚合酶链反应（PCR）成功扩增出Fv基因。大肠杆菌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.