抗CD22人源化基因工程多价微型抗体的初步表达

发布时间：2018-11-29 09:27

【摘要】： 1997年，美国FDA批准了有史以来第一个癌症治疗的裸抗体——“美罗华”嵌合抗体(Rituxan)。尽管美罗华对非霍奇金淋巴瘤(NHL)有效而且毒性可以控制，但并不是对所有的病人都有疗效，甚至所有有治疗反应的病人最后都会复发，然而美罗华激励研究者去寻找新的靶点。CD22是众多靶点中的一个，表达于正常和癌变的B细胞表面。以CD22为靶点的治疗不会影响任何不表达CD22的组织，也不会抑制新的B细胞生成。 NHL的治疗方法有放射治疗、外科治疗、生物治疗等。免疫导向治疗作为生物治疗的一种，在淋巴瘤治疗中将发挥重要作用。研究结果表明，，多价微型抗体由于抗原结合位点数目的增加而有效地提高了其亲和性，所形成的抗原-抗体复合物更为稳定，更适宜于免疫导向治疗。本实验的主要目的是探索表达抗CD22人源化基因工程多价微型抗体的方法，以期获得表达目的抗体的基因工程菌株和重组病毒株。本实验首先通过PCR分别扩增出目的基因Fc-500、CH3-1300、Fc-1600，再通过亚克隆构建分泌表达质粒pPIC9-CH3和pPIC9-Fc，电转化到巴斯德毕赤氏酵母GS115中，通过同源重组，将目的基因整合到GS115基因组中进行表达。研究表达产物，证明获得可以表达抗CD22人源化基因工程多价微型抗体的菌株GS115-CH3、GS115-Fc。将这两株酵母菌传代4次后，通过PCR检测，证明这两株菌都具有较高的稳定性。另外，构建表达质粒pAcSG2-CH3和pAcSG2-Fc，将pAcSG2-CH3质粒转染Sf9细胞，通过空斑纯化重组杆状病毒Sf9-CH3，病毒滴度为4.5×10~7pfu／ml，并进行相应的检测。结果表明，获得了在昆虫杆状病毒稳定表达抗CD22人源化基因工程多价微型抗体的重组病毒株。
[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.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;Q789

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