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[目的]制备抗乙酰胆碱受体主要免疫原区单链抗体A 7基因,为基因治疗重症肌无力奠定基础.[方法]构建含有抗乙酰胆碱受体主要免疫原区单链抗体A 7基因的重组原核表达载体pHEN 2-单链抗体A 7,将其转化至大肠杆菌HB 2151中,以异丙基--βD-硫代半乳糖苷(IPTG)为诱导剂,在外周质中进行表达,以硼酸钠裂解细菌细胞壁,制备含有单链抗体的裂解液,应用鼠抗c-myc单克隆抗体的斑点杂交试验检测表达的单链抗体A 7基因.[结果]在大肠杆菌的外周质中可见单链抗体A 7基因表达,培养上清液中未见单链抗体A 7基因表达.[结论]抗乙酰胆碱受体主要免疫原区单链抗体A 7基因在原核细胞成功表达.
[Objective] The aim of the study was to prepare single-chain antibody A 7 gene, which is the major antigen of anti-acetylcholine receptor, and lay a foundation for gene therapy for myasthenia gravis [Method] To construct a recombinant prokaryotic expression vector containing single- The expression vector pHEN 2-single chain antibody A 7 was transformed into E. coli HB 2151 and expressed in the periplasm using isopropyl-βD-thiogalactoside (IPTG) as an inducer. Sodium was used to lyse the bacterial cell wall to prepare lysis buffer containing single chain antibody, and the expressed single chain antibody A 7 gene was detected by dot blot hybridization assay using mouse anti-c-myc monoclonal antibody. [Results] Single-stranded antibody was expressed in the periplasm of E. coli Antibody A 7 gene was expressed in culture supernatant, and no single-chain antibody A 7 gene was expressed in culture supernatant. [Conclusion] A 7 gene, the major immunogen of AChR, was successfully expressed in prokaryotic cells.