重症肌无力抗乙酰胆碱受体单链抗体a7基因真核表达载体构建

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1、延边人学柏原生物学专业顺Ij学位论文摘丝重症肌无力抗乙酰胆碱受体单链抗体A7真核表达载体的构建摘要重症肌无力(myastheniagravis，MG)是抗乙酰胆碱受体(acetylcholinereceptor,AchR)抗体介导的器官特异性自身免疫病。AchR是由0【2pY6五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O【亚单位上的主要免疫原区(mainimmunogenicregion，MIR)。当致病性抗体与神经肌肉接头处突触后膜上相邻两个AchRO【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

2、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(singlechainvariablefragment，ScFv)作为单价片段，只能与一个Ct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pHEN2一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EcoRI和AvrII双酶切后用低

3、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．coliDH5c,并扩增，再用Avr1I和EcoRI酶切检查ScFvA7基因插入的证确性。构建pPIC9K-ScFvA7重组载体经测序发现核苷酸序列『F确，并且ScFvA7基因『F确克隆至载体丌放读码框架内。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。关键词：重症肌无力：单链抗体：真核表达载体延边人学绷原生物学’(％IkO)iIj学位论爻英义摘世Constructionofe

4、ukaryoticexpressionvectorofA7geneofsinglechainvariablefragmentagainstacetylcholinereceptorinmyastheniaAbstractgravlsMyastheniagravis(MG)isanorgan—specificautoimmunediseasemediatedbyautoantibodiesdirectedagainstacetylcholinereceptor(AchR)．AchRisatransmembraneglycoprotein，cons

5、istingoffivesubunitsa213y8．TheantibodiesinseraofMGpatientsaremainlythosedirectagainstthemainimmunogenicregion(MIR)onO【一subunitofAchR．ThedamageofpostsynapticmembraneandAchRlossbyantigenmodulationandactivationofcomplementresultinnmscleweakness，whenpathogenicantibodybindtoMIRof

6、consecutivetwoQ—subunitofAchRonpostsynapticmembraneinneuromuscularjunction．Asunivalentantibodyfragments，singlechainvariablefragment(ScFv)whichderivedfromantibodyagainstAchRcanbindtoonlyoneMIRofO【一subunit．thereforeitcannotcauseantigenmodulation．ItalsocannotcausethedamageofAch

7、RbyactivationcomplementbecauseofhavingnoFcfragmentHoweversuchfragmentsarecapableofprotectingthereceptoragainstthelossinducedbyintactanti——MIRandibodies．InthisexperimentthemouseScFvA7geneamplifiedbyPCRfromtherecombinantvectorspHEN2一ScFvA7waspurifiedbyWizardPCRPrepsDNAPurifcat

8、ionSystem，thendigestedwithEcoRIandAvrII．Thedigestedproductswereruninlowmelt