单链抗体A 7基因真核表达载体构建.docVIP

单链抗体A 7基因真核表达载体构建 　【摘要】 ［目的］ 构建抗乙酰胆碱受体主要免疫原区单链抗体A 7基因的真核表达载体，为单链抗体A 7基因的真核表达及基因治疗重症肌无力奠定基础. ［方法］ 应用PCR技术，从已构建的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上扩增单链抗体A 7基因并纯化，将纯化后的PCR产物经EcoRⅠ和AvrⅡ双酶切后，用低熔点琼脂糖凝胶电泳回收并纯化，再与经同样酶切并纯化的真核表达载体pPIC 9 K连接，将其产物转化至大肠杆菌DH 5 α后扩增，再用AvrⅡ和EcoRⅠ酶切和测定序列检查ScFv A 7基因插入的准确性. ［结果］ 构建pPIC 9 K-ScFv A 7重组载体，经序列测定检查核苷酸序列正确，且ScFv A 7基因准确地克隆至载体开放读码框架内. ［结论］ 成功地构建了抗乙酰胆碱受体主要免疫原区单链抗体A 7基因的真核表达载体. 【关键词】 重症肌无力；抗体；真核表达载体 ABSTRACT: OBJECTIVE To construct a recombinant eukaryoic expression vector of the single chain variable fragment (ScFv) A 7 of antibody against the main immunogenic region (MIR) of acetylcholine receptor (AChR) for the further eukaryoic expression and the gene therapy in myasthenia gravis (MG). METHODS The ScFv A 7 gene was amplified by PCR from the recombinant prokaryotic expression vector pHEN 2-ScFv A 7, then purified and digested with EcoRⅠand AvrⅡ. The digested products were run in low melting point agarose gel eletrophoresis and purified by Wizard PCR Preps DNA Purification System, then ligated into eukaryotic expression vector pPIC 9 K digested and purified by the same method. The recombinant vector pPIC 9 K-ScFv A 7 was transformed into E. coli DH 5 α for amplification and isolated and digested with AvrⅡ and EcoRⅠ again. The pPIC 9 K-ScFv A 7 gene was analysed for an insert of the right size by digestion with AvrⅡ and EcoRⅠ. RESULTS The sequencing showed that the nucleotide sequence of constructed ScFv A 7 was correct and cloned into the open reading frame (ORF) in pPIC 9 K. CONCLUSION The recombinant eukaryoic expression vector pPIC 9 K-ScFv A 7 has been successfully constructed. Key words:myasthenia gravis;antibodies;eukaryotic expression vector 重症肌无力是抗乙酰胆碱受体（acetylcholine receptor, AchR）抗体介导的器官特异性自身免疫病,AchR是由α 2 βγδ 等5个同源亚单位组成的跨膜糖蛋白［1］.现已发现,重症肌无力患者血清中2/3的抗AchR抗体针对的是α亚单位的主要免疫原区的第67～76位氨基酸表位［2］.当致病性抗体与神经肌肉接头处突触后膜上相邻2个AchR α亚单位上的主要免疫原区结合后，通过抗原调变或激活补体作用使突触后膜损伤［3］，AchR数量减少，结果出现受累肌肉收缩无力及麻痹症状［4］.由抗AchR抗体制备的单链抗体（single chain var