机构地区:[1]安徽医科大学附属省立医院神经内科,合肥230001 [2]安徽医科大学生化教研室
出 处:《中华神经科杂志》2007年第8期560-563,共4页Chinese Journal of Neurology
基 金:安徽省人才开发资金(20052041);安徽省教委自然科学基金(2005KJ343zc)
摘 要:目的克隆和表达人乙酰胆碱受体α亚基1-210(hAChR α1-210),并以该表达产物免疫 Lewis 鼠诱导实验性自身免疫性重症肌无力(EAMG)模型。方法将经逆转录-PCR 扩增出的目的基因片段 AChR α1-210在大肠杆菌中诱导表达,经 SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和 Westernblot 鉴定表达产物为 rhAChR α1-210。将凝胶上一定剂量 rhAChR α 1-210融合蛋白与完全氟氏佐剂(CFA)充分乳化后,经皮下多点注入实验组,对照组则注入等剂量与 CFA 充分混匀乳化的空菌总蛋白。观察免疫后鼠体重的变化,并给予 Lennon 临床评分;低频重复电刺激检查电衰减反应和 ELISA法检测血清 AChR-ab 滴度作为评价造模是否成功的客观依据。结果将一定量的表达产物 rhAChRα1-210融合蛋白免疫 Lewis 鼠,并在4周后强化免疫一次。在强化免疫10 d 后实验组(n=10)部分鼠出现体重下降和肌无力,至实验结束时实验组有7只鼠 Lennon 临床评分达1级以上,对照组未见体重下降和肌无力表现。低频重复电衰减检查显示实验组3 Hz 和5 Hz 衰减率分别为13.90%±4.20%、13.20%±2.62%,对照组则为5.60%±2.06%、7.70%±2.40%,两组比较差异有统计学意义。ELISA 法检测血清 AChR-ab 滴度结果表明实验组阳性率为70%,对照组未出现阳性,两组比较差异有统计学意义。结论采用 rhAChR α1-210融合蛋白可成功诱导 EAMG 动物模型,与经典方法相比具有操作方法简单、成本较低、免疫原充足等优点。Objective To induce experimental autoimmune myasthenia gravis (EAMG) in Lewis rats using human AChR α-subunit 1-210( hAchR α1-210) obtained by cloning and expression technique. Methods hAChR α1-210 cDNA was amplified by RT-PCR and expressed in E coli. The target protein was confirmed by SDS-PAGE and Western blot. The experimental group was intradermally injected by the fusion protein emulsified in CFA and boosted, and the control group was injected by the expressed product of empty plasmid. The clinical manifestation was evaluated by measuring the body weight and Lennon clinical score. Disease was further confirmed by low frequency repetitive nerve stimulation for positive decremental response and ELISA assay for serum AChR-ab titers. Results hAChR α1-210 was cloned and expressed successfully. The female Lewis rats were immunized by the fusion protein as an emulsion in CFA and boosted after 4 weeks. Clinical signs of MG including loss of body weight and weakness become evident 10 days after the second immunization in some experimental rats. Positive clinical score was seen in 70% immunized rats at the end of the experiments, and none of the control rats did not develop any clinical signs. The electric decrement of 3,5 Hz by low frequence repetitive nerve stimulation were 13.90% ± 4. 20%, 13.20% ± 2. 62% in the experimental group, 5.60% ±2. 06% ,7.70% ±2.40% in the control group, and there was significant difference between them. Positive ratio of serum AChR-ab titers by ELISA was 70% in the experimental group, none in the control group, and the difference was significant. Our data suggest that 7 out of 10 of the experimental rats were induced into the EAMG models, while none sick were found in control rats. Conclusions The expressed hAChR α1-210 can induce EAMG animal model. The method is simpler and cheaper. The immunogen is more sufficient compared with those of classic EAMG models.
关 键 词:重症肌无力 实验性自身免疫性 受体 胆碱能 重组融合蛋白质类
分 类 号:R746.1[医药卫生—神经病学与精神病学]
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