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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 clone and express the human subunit of human cholinergic receptor α - 110 (hAChR α1-210) and induce Lewis rats to induce experimental autoimmune myasthenia gravis (EAMG). Methods The gene fragment of AChR α1-210 amplified by reverse transcription-PCR was induced in E. coli. The expressed product was identified as rhAChR α1-210 by SDS-PAGE and Western blot. The gel was a certain dose of rhAChR α 1-210 fusion protein and complete Freund’s adjuvant (CFA) emulsified, the experimental group was injected subcutaneously multi-point, while the control group was injected with a uniform dose of CFA fully emulsified emulsification of total bacteria protein. The changes of body weight after immunization were observed and the Lennon clinical score was given. The electric decay reaction was examined by low-frequency repetitive electrical stimulation and the serum AChR-ab titer was determined by ELISA as an objective basis to evaluate the success of modeling. Results A certain amount of rhAChRα1-210 fusion protein was immunized with Lewis rats and boosted once after 4 weeks. In the experimental group (n = 10), mice in the experimental group (n = 10) ten days after the booster immunization showed decreased body weight and muscle weakness. By the end of the experiment, Lennon’s clinical score of seven mice in the experimental group reached a grade of 1 or higher, and no weight loss and weakness manifested in the control group . Low-frequency repetitive electrical decay examination showed that the decay rates of 3 Hz and 5 Hz in experimental group were 13.90% ± 4.20% and 13.20% ± 2.62% respectively, while those in control group were 5.60% ± 2.06% and 7.70% ± 2.40%, respectively Statistical significance. The results of ELISA showed that the positive rate of AChR-ab in serum was 70% in experimental group, but not in control group. There was significant difference between the two groups. Conclusion The rhAChR α1-210 fusion protein can induce the EAMG animal model successfully. Compared with the classical method, the method has the advantages of simple operation method, lower cost and sufficient immunogen.