摘要
目的为乙酰胆碱毒蕈碱(M)受体亚型特异性的变构调节剂及基因工程的研究提供实验平台。方法用PCR及搭桥PCR法对乙酰胆碱M2及M5受体作以下突变:①将N-糖基化位点Asp突变为Asn;②删除对蛋白酶敏感的M受体的第三个细胞内环;③在C端添加凝血酶识别位点(CMV)和6-His标记。将PCR扩增出重组嵌合蛋白基因亚克隆到杆状病毒转移载体,制备重组杆状病毒并感染昆虫细胞表达M2/M5受体蛋白。Western印迹及放射性配体受体结合实验验证受体的正确表达及功能。结果通过搭桥PCR,成功扩增出1018 bp的重组M2受体和1041 bp重组M5受体核酸序列;使用pUC/M13的扩增引物成功构建M2/M5重组转移载体。将重组载体质粒与线性化病毒DNA共转染昆虫细胞Sf9,制备重组杆状病毒并感染昆虫细胞,见细胞空泡样病变。Western印迹分析确定重组杆状病毒感染昆虫细胞M2/M5蛋白表达,放射性配体受体饱和实验结果表明,表达的重组受体蛋白与[3H]N-甲基-东莨菪碱具有特异性结合能力。结论 Sf9昆虫细胞能够表达M2及M5重组受体蛋白,M2及M5重组受体蛋白的病毒样颗粒可用于M受体的新药研究。
OBJECTIVE To study the expression of human muscarinic receptors （ M2 and M5 recombinant receptors in the baculovirus expression system. METHODS The mutation of human wild type M2 and M5 receptors was constructed by PCR or/and overlap PCR as follows： ① The putative glycosylation residues Asp 2, 3, 6, and 9 were replaced with Ash to prevent molecular heterogeneity ; ② The central part of the protease-susceptible third intracellular loop was deleted; ③ A hexa-histidine tag and a thrombin cleavage site were added at the C terminus for purification. The recombinant receptor gene was confirmed and amplified by PCR, and subcloned to baculovrius pFastBac 1 vector. Then the recombinant vector was co-transfected with the linearized virus DNA into sf9 cells by Lipofectamine. The recombinant M2 and M5 receptor protein was prepared and purified. The expression level of M2 and M5 receptors was evaluated by Western blotting, and pharmacological characteristics were confirmed by radio-legend binding assay. RESULTS The target DNA fragment of M2（ 1018 bp） and M5 （1041 bp） recombinant receptors was amplified by overlap PCR. The recombinant plasmid pfastbacl/M2 （M5 ） vector was successfully constructed, and transfected to Sf9. Vacuolus pathological changes were observed within ceils compared to non-transfection of Sf9. The baculovirus particle protein was prepared and purified from these infected cells. The expression of M2/M5 was further confirmed by Western blotting. The specific binding character of recombinant M2/M5 receptors was detected by radio-legend binding assay. CONCLUSION The expression of M2 and M5 recombinant receptors in the baculovirus expression system will facilitate studies on new drugs from M receptor or genetic engineering.
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2011年第3期320-326,共7页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然科学基金资助项目(30672445)~~
