日本血吸虫酚氧化酶天然蛋白分子特征的初步分析  

PRELIMINARY STUDIES ON MOLECULAR CHARACTER OF NATURAL PHENOL OXIDASE PROTEIN IN SCHISTOSOMA JAPONICUM

作　　者：蔡国斌[1] 何立[1] 蒋明森[1] 章莹[1] 赵琴平[1] 杨孟祥[1] 

机构地区：[1]武汉大学医学院人体寄生虫学教研室血吸虫病研究室,武汉430071

出　　处：《中国血吸虫病防治杂志》2004年第2期115-117,共3页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金专项基金项目 ( NO.3 0 2 40 0 74);湖北省自然科学基金项目 ( No.99J16);湖北省教育厅资助项目 ( No.2 0 0 1A14 0 0 9)

摘　　要：目的　初步分析日本血吸虫酚氧化酶 (phenol oxidase,PO)天然蛋白分子的分子量及其亚基组成。方法　将 4 2 d龄活成虫置于含 0 .0 5 %戊巴比妥钠的 RPMI16 4 0培养基中 2 3℃孵育 8h后 ,PBS(p H6 .8)洗净 ,分离雌、雄成虫 ,研磨呈匀浆 ,超声粉碎、低温高速离心 ,取上清 (含 PO)进行聚丙烯酰胺凝胶电泳 (PAGE) ,分别在两侧切取胶宽约 1cm进行酶染色 ,然后用解剖刀准确切下相应的未染色的凝胶。经真空冷冻干燥机冻干后碾成粉末 ,高速电动匀浆成浆糊状 ,冷浸过夜 ,高速离心得上清即为日本血吸虫酚氧化酶粗蛋白 ,进行 SDS- PAGE。利用 SDS- PAGE与 PAGE(点加 1孔预染 Mark蛋白 )的结果 ,对照分析酚氧化酶蛋白的可能分子量及其亚基组成。结果　 PAGE:日本血吸虫雌虫、雄虫酚氧化酶蛋白均表现为迁移率相同的 1条主带 ,其对应分子量大约为 81k D。SDS-PAGE:酚氧化酶粗蛋白共出现 5条带 ,分别为 81、6 4、5 4、4 1k D和 2 7k D。结论　初步分析日本血吸虫雌、雄成虫酚氧化酶天然蛋白分子的分子量及其亚基组成基本相同 ,可能为单体酶 (分子量 81k D)或由 3个 2 7k D相同亚基构成的同聚体。Objective To analyse the molecular weight and the subunit of natural phenol oxidase protein in Schistosoma japonicum. Methods Living adult worms (aged 42 d) were incubated in RPMI 1640 containing 0 05% sodium phenobarbital for 8 hours at 23℃. Then female worms and male worms were separated, collected and treated by homogenating, ultrasound fragmentation and high speed centrifugation respectively.Supernatant fractions (containing phenol oxidase activities) were obtained. They were analyzed by means of polyacrylamide gel electrophoresis (PAGE). The phenol oxidase rude protein was obtained by exactly cutting the corresponding gel position of PO activities with scalpel. The PO molecular character was analysed by means of SDS-PAGE and previous PAGE (plus a stained Mark). Results PAGE: The similarity of zymogram patterns of phenol oxidase in Schistosoma japonicum from females and males were observed, including a major band and its same migrating. The corresponding molecular weight was about 81 kD. SDS-PAGE: Phenol oxidase rude protein had 5 bands: 81?64?54?41 kD and 27 kD? Conclusion The natural phenol oxidase protein′s molecular weight and the subunit were same both in female and in male Schistosoma japonicum and the PO probably was a mono-enzyme (MW81 kD) or a homopolymer consisting of three 27 kD subunits by primary analysis. The exactly molecular character of PO waits on more researching.

关 键 词：日本血吸虫 酚氧化酶 分子量 亚基组成 

分 类 号：R383.24[医药卫生—医学寄生虫学]