日本血吸虫成虫蛋白质的性别差异性研究

目的 研究日本血吸虫成虫蛋白质表达的性别差异性。方法 利用双向电泳技术对日本血吸虫中国大陆株雌雄虫体蛋白质进行电泳分析。结果 在 43kDa ,pI 5 .6 0～ 5 90处 ,雄虫有一条长度和宽度均超过雌虫并由多个斑点连在一起组成的条带 ,并有 3个较雌虫大的斑点 ;雌虫有 7个特异性斑点 ,且有一处较雄虫深的着色区。结论 血吸虫成虫蛋白质表达有性别差异性。

日本血吸虫虫卵、童虫和雌雄成虫膜蛋白的双向电泳

Membrane proteins were extracted from eggs, schistosomulum, adult male and female worms of Schistosoma japonicum in order to analyze the differently expressed profile by two dimensional electrophoresis. Schistosomulum and adult worms were obtained from rabbits infected with 1 500 cercariaes on 14 and 42 days after challenge, respectively. Adult male and female worms on 42 days were manually detached and stored into liquid nitrogen until use. Eggs were collected by Percoll TM from the liver of rabbits. ProteoPrep Membrane Extraction Kit TM was employed to extracted membrane proteins by reducing and alkylating with TBP and iodoacetamide from 200 mg of eggs, schistosomulums, adult male worm and female worms, respectively. Immobilized pH gradient strips with a linear pH range of 3-10 (130 mm) were rehydrated together with membrane proteins (30 μg) in 250 μl solution containing 7 mol urea, 2 mol thiourea, 2% SB3-10, 4% CHAPS, 40 mmol Tris, 30 mmol DTT, then separated on 12.5% SDS polyacrylamide gel for the second dimensional electrophoresis. Gels were stained with silver, scanned by Labscan, and analyzed using ImageMaster TM Analysis software. The 2D maps of egg, schistosomulum, adult female worm and male worm were showed 78±3、67±3、108±4 and 122±4 spots respectively. There were 35±1 spots which showed specific expression in female worm as compared with male worm, but 45±2 spots were in male worms. Most differently expressed spots between male and female worms were located in the area of 40-70 kD and pI 4-7. The large number of unique spots from schistosomulum was located in the area of alkalescence. The 2D map of for adult male worms uniquely showed 5 spots as compared with that of schistosomulum and female worm. The female worm showed 4 unique spots as compared with that of schistosomulum, egg and male worm. The unique spots between male and female worms were identified by the database of SWISS 2D-PAGE according to the molecular weight and isoelectronic point. Calreticulin 1, methyltransferase, outer membrane protein tolc and oxygen-evolving enhancer protein 1-1 were uniquely showed in adult male worm after pairing. On the other hand, enolase, outer membrane protein X, ferrienterobactin receptor and heat shock cognate 70 kD protein 3 were uniquely showed in adult female worm. In conclusion, there are different expressions of membrane proteins from egg, schistosomulum, adult male worm and female worms of Schistosoma japonicum. These proteins, which were uniquely expressed between adult male and female worms after pairing, were involved in signal transduction and metabolism

日本血吸虫生殖相关抗原31～32kDa分子的质谱分析与鉴定

目的利用蛋白质组学技术分离、鉴定日本血吸虫天然分子31～32kDa抗原组分。方法收集感染后32d的日本血吸虫成虫,制备成虫蛋白,经一维电泳、固相pH梯度双向凝胶电泳分离,选取31～32kDa附近的蛋白点进行质谱分析鉴定,通过凝胶图像分析软件进行结果比较。结果二维电泳结果显示31～32kDa附近有13个蛋白点,经MALDI-TOF-MS鉴定及分析,发现3个蛋白分子分别与曼氏血吸虫Actine-2、日本血吸虫3-磷酸甘油脱氢酶、曼氏血吸虫组织蛋白酶B肽链内切酶有高度的同源性。结论31～32kDa组分中含有这3个蛋白点,它们与日本血吸虫31～32kDa天然分子中起保护性的有效分子密切相关。同时,它们的发现也为天然分子疫苗向基因工程疫苗的推进奠定了基础。

紫外线照射前后日本血吸虫尾蚴可溶性虫体蛋白组分的比较

目的利用蛋白质组学技术分离、鉴定日本血吸虫正常尾蚴及紫外线致弱尾蚴虫体间差异表达蛋白。方法分别收集与制备血吸虫正常尾蚴和致弱尾蚴虫体总蛋白,经固相pH梯度双向凝胶电泳分离。凝胶银染并利用ImageMaster2DSoftware5.0凝胶图像分析软件进行比较分析,选取差异表达蛋白点经基质辅助激光解析离子飞行时间质谱仪进行鉴定。结果二维凝胶电泳图像分析结果显示:正常尾蚴和致弱尾蚴各分离出至少1277、1173个清晰、独立的蛋白点,蛋白点匹配率为72.7%,大多数蛋白点相对分子量处于22000～95000范围内,理论等电点为5～8。尾蚴经紫外线致弱前后共发现18个差异表达蛋白,其中5个蛋白于致弱尾蚴中表达消失,12个表达下调,1个表达增强,未见新增表达蛋白。其中13个差异表达蛋白经MALDI-TOF-MS鉴定分析,获悉其肽指纹图谱、理论等电点、分子量及表达水平变化等相关信息。结论利用二维电泳、MALDI-TOF-MS等蛋白质组学技术,共鉴定出13个致弱尾蚴差异表达蛋白,为进一步阐明致弱尾蚴诱导宿主产生高免疫保护力的分子机制提供了实验基础。

日本血吸虫成虫性别差异蛋白的筛选及鉴定

目的筛选和鉴定日本血吸虫成虫性别差异蛋白。方法自日本血吸虫（Schistosoma japonicum，S．japonicum）尾蚴感染的2只家兔颈动脉灌注生理盐水冲虫，分别收集雌雄成虫各约200条。提取雌、雄成虫的水溶性和脂溶性蛋白各300μg，采用双向凝胶电泳技术分别进行分离。银染后扫描成像，用Image Master Platinum 2D 5．0软件进行分析，筛选差异蛋白，并予质谱鉴定。结果雌虫和雄虫的水溶性蛋白分别检出（255±10）、（224±12）个蛋白点，疏水蛋白分别检出（200±11）、（132±8）个蛋白点；鉴定的6个差异蛋白质中，5个雌性表达上调的差异蛋白为硫氧还蛋白，铁蛋白-1重链，泛素结合酶样蛋白Mms 2-泛素复合体可溶性结构B链，热休克蛋白10和胞浆脂肪酸结合蛋白突变体H，1个雄性表达上调的差异蛋白为48kDa组胺受体亚基肽4。结论获得了日本血吸虫成虫性别差异蛋白质。