本文選題：重癥肌無(wú)力 + 單鏈抗體　； 參考：《延邊大學(xué)》2006年碩士論文

【摘要】：重癥肌無(wú)力(myasthenia gravis，MG)是抗乙酰膽堿受體(acetylcholine receptor，AchR)抗體介導(dǎo)的器官特異性自身免疫病。AchR是由α_2β_γδ五個(gè)同源亞單位組成的跨膜糖蛋白，MG患者血清中多數(shù)抗AchR抗體針對(duì)的是α亞單位上的主要免疫原區(qū)(main immunogenic region，MIR)。當(dāng)致病性抗體與神經(jīng)肌肉接頭處突觸后膜上相鄰兩個(gè)AchR α亞單位上的MIR相結(jié)合后，通過(guò)抗原調(diào)變或激活補(bǔ)體作用使突觸后膜損傷，AchR數(shù)量減少，，結(jié)果出現(xiàn)肌肉收縮無(wú)力。由抗AchR抗體制備的單鏈抗體(single chain variable fragment，ScFv)作為單價(jià)片段，只能與一個(gè)α亞單位上的MIR結(jié)合，因此不引起抗原調(diào)變作用，且ScFv無(wú)Fc段，不能激活補(bǔ)體導(dǎo)致AchR損傷；但是，ScFv與MIR結(jié)合后，可以特異性地封閉抗AchR抗體的結(jié)合位點(diǎn)，從而對(duì)AchR起到保護(hù)作用。 本實(shí)驗(yàn)應(yīng)用聚合酶鏈反應(yīng)(PCR)方法從重組質(zhì)粒pHEN2-ScFvA7上擴(kuò)增出鼠源性ScFvA7基因，應(yīng)用純化試劑盒進(jìn)行純化。純化后的PCR產(chǎn)物經(jīng)EcoR Ⅰ和AvrⅡ雙酶切后用低熔點(diǎn)瓊脂糖凝膠電泳回收并純化，再與經(jīng)同樣酶切并純化的真核表達(dá)載體pPIC9K連接。將連接產(chǎn)物轉(zhuǎn)化E．coli DH5α并擴(kuò)增，再用AvrⅡ和EcoR Ⅰ酶切檢查ScFvA7基因插入的正確性。構(gòu)建pPIC9K-ScFvA7重組載體經(jīng)測(cè)序發(fā)現(xiàn)核苷酸序列正確，并且ScFvA7基因正確克隆至載體開放讀碼框架內(nèi)。結(jié)果表明已成功的構(gòu)建了pPIC9K-ScFvA7重組表達(dá)載體，為下一步進(jìn)行真核表達(dá)及之后對(duì)MG的基因治療奠定了基礎(chǔ)。
[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 偽 2 尾 _ 緯 未. Most of the anti AchR antibodies are directed against 偽 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 偽 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 偽 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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