【摘要】： 1997年，美國FDA批準(zhǔn)了有史以來第一個(gè)癌癥治療的裸抗體——“美羅華”嵌合抗體(Rituxan)。盡管美羅華對非霍奇金淋巴瘤(NHL)有效而且毒性可以控制，但并不是對所有的病人都有療效，甚至所有有治療反應(yīng)的病人最后都會復(fù)發(fā)，然而美羅華激勵(lì)研究者去尋找新的靶點(diǎn)。CD22是眾多靶點(diǎn)中的一個(gè)，表達(dá)于正常和癌變的B細(xì)胞表面。以CD22為靶點(diǎn)的治療不會影響任何不表達(dá)CD22的組織，也不會抑制新的B細(xì)胞生成。 NHL的治療方法有放射治療、外科治療、生物治療等。免疫導(dǎo)向治療作為生物治療的一種，在淋巴瘤治療中將發(fā)揮重要作用。研究結(jié)果表明，，多價(jià)微型抗體由于抗原結(jié)合位點(diǎn)數(shù)目的增加而有效地提高了其親和性，所形成的抗原-抗體復(fù)合物更為穩(wěn)定，更適宜于免疫導(dǎo)向治療。 本實(shí)驗(yàn)的主要目的是探索表達(dá)抗CD22人源化基因工程多價(jià)微型抗體的方法，以期獲得表達(dá)目的抗體的基因工程菌株和重組病毒株。 本實(shí)驗(yàn)首先通過PCR分別擴(kuò)增出目的基因Fc-500、CH3-1300、Fc-1600，再通過亞克隆構(gòu)建分泌表達(dá)質(zhì)粒pPIC9-CH3和pPIC9-Fc，電轉(zhuǎn)化到巴斯德畢赤氏酵母GS115中，通過同源重組，將目的基因整合到GS115基因組中進(jìn)行表達(dá)。研究表達(dá)產(chǎn)物，證明獲得可以表達(dá)抗CD22人源化基因工程多價(jià)微型抗體的菌株GS115-CH3、GS115-Fc。將這兩株酵母菌傳代4次后，通過PCR檢測，證明這兩株菌都具有較高的穩(wěn)定性。 另外，構(gòu)建表達(dá)質(zhì)粒pAcSG2-CH3和pAcSG2-Fc，將pAcSG2-CH3質(zhì)粒轉(zhuǎn)染Sf9細(xì)胞，通過空斑純化重組桿狀病毒Sf9-CH3，病毒滴度為4.5×10~7pfu／ml，并進(jìn)行相應(yīng)的檢測。結(jié)果表明，獲得了在昆蟲桿狀病毒穩(wěn)定表達(dá)抗CD22人源化基因工程多價(jià)微型抗體的重組病毒株。
[Abstract]:In 1997, the United States FDA approved the first ever nude antibody for cancer treatment, a chimeric antibody (Rituxan). Although Merlot is effective and toxic to non-Hodgkin 's lymphoma, it does not work in all patients, and even all patients who respond to the treatment end up relapsing. CD22 is one of many targets expressed on the surface of normal and cancerous B cells. Treatment targeting CD22 does not affect any tissue that does not express CD22, nor does it inhibit the production of new B cells. The treatment methods of NHL include radiotherapy, surgical treatment, biological therapy and so on. Immunoreactive therapy, as a biotherapy, will play an important role in the treatment of lymphoma. The results show that the multivalent microantibodies can effectively improve their affinity due to the increase of the number of antigen-binding sites, and the antigen-antibody complexes are more stable and more suitable for immunoreactive therapy. The main purpose of this experiment is to explore the method of expressing polyvalent microantibodies against CD22 humanized genetic engineering in order to obtain genetically engineered strains and recombinant virus strains expressing the target antibody. In this experiment, the target gene Fc-500,CH3-1300,Fc-1600, was amplified by PCR and then subcloned to construct the secretory expression plasmids pPIC9-CH3 and pPIC9-Fc, into Pichia pastoris GS115 by homologous recombination. The target gene was integrated into the GS115 genome for expression. The expression product was studied. It was proved that the strain GS115-CH3,GS115-Fc. could express polyvalent microantibodies against human genetic engineering of CD22. After the two yeast strains were subcultured for 4 times, the stability of the two strains was proved by PCR detection. In addition, the expression plasmids pAcSG2-CH3 and pAcSG2-Fc, were constructed to transfect the pAcSG2-CH3 plasmid into Sf9 cells. The recombinant baculovirus Sf9-CH3, virus titer was 4.5 脳 10 ~ (7) pFu / ml, and the corresponding detection was carried out. The results showed that the recombinant virus strain which stably expressed polyvalent microantibodies against CD22 humanized gene engineering in insect baculovirus was obtained.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392;Q789

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本文編號：2364620