曼氏无针乌贼(Sepiella maindroni)血蓝蛋白酚氧化酶样活性研究和相关功能单元片段的克隆

STUDIES ON PHENOLOXIDASE-LIKE ACTIVITY OF HEMOCYANINS AND cDNA CLONING OF RELATED FUNCTIONAL UNIT IN SEPIELLA MAINDRONI

采用分光光度法对曼氏无针乌贼血蓝蛋白及相关功能单元(FUs)的酚氧化酶活性进行研究,并获得该功能单元的序列信息。结果表明,曼氏无针乌贼血蓝蛋白能氧化左旋多巴和邻苯二酚而不能氧化酪氨酸单酚,说明该血蓝蛋白可能具有酚氧化酶活性,并属于儿茶酚酶类;进一步研究显示,曼氏无针乌贼血蓝蛋白酶解片段FUg的酶活特性显著区别于其它功能单元,并与天然血蓝蛋白的酶活变化相一致,经同源克隆得到FUg可能的蛋白质编码区(CDS)区包含969个核苷酸,编码323个氨基酸,序列比对结果显示,该序列与商乌贼(Sepia officinalis)和水蛸(Octopus dofleini)血蓝蛋白有很高的同源性。

Hemocyanins （Hcs） from haemolymph of the cuttlefish, Sepiella maindroni were studied by spectrophotometric method and a method for determination of phenoloxidase-like activity of related functional units was established and its eDNA cloning was also obtained. The results showed that L-DOPA and catechol had higher affinity with PO than tyramine which suggested that it belonged to the catehcol oxdiase-type. It was found that, comparing with other fragments and functional units exhibited phenoloxidase-like activity with L-DOPA as substrate, FUg was showed to be much better not only in intrinsic but also in proteolytically induced activity for the further research. Partial cDNA of FUg with possible CDS was cloned through homologous cloning method, which consisted of 969bp with encoded 323 amino acids. There were very high identities in eDNA cloning from FUg between this sequence of Sepia officinalis and Octopus dofleini.