【作者】 金桂花；

【导师】 孟繁平；

【作者基本信息】 延边大学 ， 病原生物学， 2003， 硕士

【摘要】 重症肌无力（myasthenia gravis , MG）患者血清中2/3的抗乙酰胆碱受体（acetylcholine receptor, AChR）抗体是针对α亚单位上主要免疫原区（main immunogenic region, MIR）的抗体，因此针对AChR MIR的抗体成为MG致病机制中的主要的致病性抗体。由抗AChR MIR抗体制备的单链抗体（single chain variable fragment, ScFv）做为单价片段既不与AChR交叉结合，也不引起补体经典途径的激活，因此不会引起AChR的丢失。但是，ScFv与MIR结合后能特异性地封闭致病性AChR抗体与AChR MIR结合，从而对AChR起到保护作用。本文应用PCR从抗AChR MIR抗体扩增重链可变区（heavy chain variable region , VH）基因，应用纯化试剂盒对PCR产物进行纯化。纯化后的VH PCR产物经NcoⅠ和 XhoⅠ酶切、低熔点琼脂糖凝胶电泳回收及纯化后与经同样酶切割并纯化的载体质粒pHEN2连接。将连接物转化E.coli DH5α扩增，分离纯化重组子再经NcoⅠ和XhoⅠ酶切，并用琼脂糖凝胶电泳检查VH基因的正确性。采用同样的方法将轻链可变区（light chain variable region , VL）基因克隆至pHEN2-VH，并转化E.coli HB2151扩增，用ApalⅠ和NotⅠ酶切检查VL基因的正确性。构建的ScFv经测序发现核苷酸序列正确，并且VH和VL基因正确克隆至载体开放读码框架内。已成功地构建抗AChR MIR ScFv基因，为进一步制备基因工程抗体奠定了基础。更多还原

【Abstract】 Antibody competition experiments suggest that about 2/3 of the antibodies in the sera of myasthenia gravis (MG) patients are directed against the main immunogenic region (MIR) of acetylcholine receptor (AChR). Univalent antibody fragments, such as single chain variable fragment (ScFv), do not cross-link AChR molecules or bind complement, and therefore do not cause AChR loss; on the contrary, such fragments derived from anti-MIR monoclonal antibodies (mAbs) seem capable of protecting the receptor against the loss induced by intact anti-MIR mAb or MG sera.The heavy chain variable region (VH) gene of a mAb against AChR amplified by polymerase chain reaction (PCR) was purified by Wizard PCR Preps DNA Purification System, then digested with NcoⅠand XhoⅠ. The digested products were run in low gelling temperature agarose eletrophoresis and purified by Wizard PCR Preps DNA Purification System, then ligated into plasmid pHEN2 digested and purified by the same method. There recombinant pHEN2-VH were transformed into E.coli DH5αfor amplification and isolated from E.coli DH5αand digested with NcoⅠand XhoⅠagain. The light chain variable region (VL) gene of the mAb was cloned to pHEN2-VH by the same ways and than the recombinant plasmids were transformed into E.coli HB2151. The VL gene was analysed for an insert of the right size by digestion with ApalⅠand <WP=5>NotⅠ. The sequencing showed that the nucleotide sequence of cons-tructed ScFv was correct and cloned into the open reading frame (ORF) in pHEN2. A ScFv gene against the MIR of AChR has been successfully constructed.更多还原