猪囊尾蚴谷胱甘肽S-转移酶的表达研究  

Expression of glutathione S-transferase in Taenia solium cysticercus

作　　者：方文[1] 肖靓靓[2] 包怀恩[2] 牟荣[2] 

机构地区：[1]贵阳医学院医学检验系,贵阳550004 [2]贵阳医学院寄生虫学教研室,贵阳550004

出　　处：《中国人兽共患病学报》2011年第5期398-402,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(No.30660168)资助

摘　　要：目的应用免疫组化、双向电泳（2-DE）结合蛋白质印迹（Western-blotting）技术分析猪囊尾蚴谷胱甘肽S-转移酶（GST）的表达。方法用猪带绦虫卵1 mL（8万个/mL）灌胃20d龄健康乳猪6头,于感染后40d、80d和120 d分别宰杀2头猪并取含囊尾蚴的肌肉及肝脏（120 d除外）制作4μm厚石蜡切片,采用Envision二步法,观察不同时期寄生在肌肉及肝脏的猪囊尾蚴GST的表达;同时制备感染后40d取自肌肉的猪囊尾蚴蛋白进行2-DE分析,将凝胶蛋白斑点转移至聚偏氟乙烯膜（PVDF膜）,用本课题组自制的大鼠抗猪带绦虫GST血清作为一抗、健康大鼠血清作为阴性对照进行western-blotting分析。结果不同时期寄生在肌肉及肝脏的猪囊尾蚴均表达GST,随感染时间增加,寄生在肌肉的猪囊尾蚴GST的表达变化不大（P〉0.05）,寄生在肝脏的猪囊尾蚴GST的表达升高（P〈0.05）;双向电泳凝胶共检测到207±9个蛋白质斑点,相对分子质量（Mr）为14 400-94 000,等电点（pI）为3.0-10.0。Western-blotting分析显示,实验组特异性抗原抗体阳性杂交斑点为1个,阴性对照未见阳性杂交斑点。将Western-blotting检测的抗原抗体阳性杂交斑点与原双向电泳凝胶斑点进行比对,找到对应蛋白斑点,经ImageMaster 2D Platinum 5.0软件分析后初步确定该蛋白斑点的pI/Mr为6.6/25 548,与猪带绦虫GST的理论推导值接近。结论感染时间及感染部位组织学特征不同,猪囊尾蚴GST的表达略有差异。Glutathione S-transferase（GST） was detected with immunohistochemistry methods,two dimensional electrophoresis and western blotting to explore the expression of GST in cysticercus cellulosae.Six piglets of 20-day-old were infected with a dose of 80 000 eggs of T.sodium and the immunohistochemistry have been done on paraffin section of cysticercus cellulosae acquired on 40,80 and 120 days after infection from muscle and liver（except 120 days）.The total protein of cysticercus cellulosae from muscle acquired 40 days after infection was separated by two-dimensional electrophoresis,and the western blotting was performed to find out GST after the protein spots on the gel were transferred to PVDF membrane.The results showed that there was GST expressing in the head and wall of the cysticercus cellulosae from different tissues at different times.As the time passed,the GST expression of cysticercus celluosae from muscle were little changed（P〉0.05）,while the GST expression of cysticercus celluosae from brain were slightly higher（P〈0.05）.There were 207±9 protein spots of cysticercus cellulosae in 2-DE gel stained by coomassie brilliant blue,and the ranges of Mr and pI were 14 400-94 000 and 3.0-10.0,respectively.Western blotting showed 1 specific antigen spots with anti-GST mixed sera of the experimental rats and none specific spot with the mixed sera of the healthy rats.The spot in the western blotting was corresponding to the protein spot in 2-DE gel.The pI/Mr of the protein in 2-DE gel was similar to the theoretical value of GST of Taenia solium.The results indicate that there is a little difference on GST expression in cysticercus cellulosae parasited in different tissues at different times.

关 键 词：猪带绦虫 囊尾蚴 谷胱甘肽S-转移酶 免疫组化 双向电泳 蛋白质印迹 

分 类 号：R383.2[医药卫生—医学寄生虫学]