重症肌无力抗乙酰胆碱受体单链抗体A7基因真核表达载体的构建

发布时间：2018-05-02 22:29

  本文选题：重症肌无力 + 单链抗体　； 参考：《延边大学》2006年硕士论文

【摘要】：重症肌无力(myasthenia gravis，MG)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_2β_γδ五个同源亚单位组成的跨膜糖蛋白，MG患者血清中多数抗AchR抗体针对的是α亚单位上的主要免疫原区(main immunogenic region，MIR)。当致病性抗体与神经肌肉接头处突触后膜上相邻两个AchR α亚单位上的MIR相结合后，通过抗原调变或激活补体作用使突触后膜损伤，AchR数量减少，，结果出现肌肉收缩无力。由抗AchR抗体制备的单链抗体(single chain variable fragment，ScFv)作为单价片段，只能与一个α亚单位上的MIR结合，因此不引起抗原调变作用，且ScFv无Fc段，不能激活补体导致AchR损伤；但是，ScFv与MIR结合后，可以特异性地封闭抗AchR抗体的结合位点，从而对AchR起到保护作用。 本实验应用聚合酶链反应(PCR)方法从重组质粒pHEN2-ScFvA7上扩增出鼠源性ScFvA7基因，应用纯化试剂盒进行纯化。纯化后的PCR产物经EcoR Ⅰ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pPIC9K连接。将连接产物转化E．coli DH5α并扩增，再用AvrⅡ和EcoR Ⅰ酶切检查ScFvA7基因插入的正确性。构建pPIC9K-ScFvA7重组载体经测序发现核苷酸序列正确，并且ScFvA7基因正确克隆至载体开放读码框架内。结果表明已成功的构建了pPIC9K-ScFvA7重组表达载体，为下一步进行真核表达及之后对MG的基因治疗奠定了基础。
[Abstract]:Myasthenia gravis (MG) is an organ-specific autoimmune disease mediated by anti-acetylcholine receptor AchR.AchR is a transmembrane glycoprotein (MG) composed of five homologous subunits of 伪 2 尾 _ 纬 未. Most of the anti AchR antibodies are directed against 伪 subunit in the serum of patients with myasthenia gravis. Main immunogenic region. When the pathogenic antibody combined with the MIR of the two adjacent AchR 伪 subunits on the postsynaptic membrane of the neuromuscular junction, the number of MIR in the injured postsynaptic membrane was reduced by antigenic modulation or activation of complement, resulting in muscle contraction weakness. Single chain variable fragment (scFv), which is prepared from anti AchR antibody, can only bind to MIR on one 伪 subunit, so it does not cause antigenic modulation, and ScFv does not have FC segment and can not activate complement to cause AchR damage, but scFv binds to MIR. It can specifically block the binding sites of anti-AchR antibody and thus protect AchR. Mouse ScFvA7 gene was amplified from recombinant plasmid pHEN2-ScFvA7 by polymerase chain reaction (PCR) and purified by purification kit. The purified PCR product was digested by EcoR 鈪

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