抗hTNF-α单克隆抗体的制备及其抗体基因克隆研究

发布时间：2018-01-06 10:21

  本文关键词：抗hTNF-α单克隆抗体的制备及其抗体基因克隆研究　出处：《中国协和医科大学》2010年硕士论文　论文类型：学位论文

  更多相关文章： 肿瘤坏死因子(TNF-α) hTNF-α抑制剂 自身免疫性疾病

【摘要】： 肿瘤坏死因子a(Tumor necrosis factor a, TNFa)是一种由活化的单核/巨噬细胞分泌的多功能细胞因子,研究表明：适量的TNFa可以协助宿主抵抗微生物入侵、杀伤某些肿瘤细胞或使体内肿瘤组织发生出血坏死,但过量的TNFa与其它炎性因子一起产生多种病理性损伤,可以引起组织损伤、重症感染性休克、自身免疫性疾病等。可以说TNF-a作为一个调节生理平衡的关键细胞因子,在人类对抗肿瘤及其他疾病中扮演了“双刃剑”的角色。 通过抑制TNFa的活性作用可以对与TNFa有关的疾病产生治疗作用,目前主要用于类风湿关节炎、抑制肿瘤的发生、生长和转移以及对多药耐药的治疗。以TNFa为靶点开发的药物包括有很多,这些药物从不同层次不同水平抑制TNFa的产生和发生作用从而达到抗TNFa治疗的目的。临床研究表明抗TNFa治疗的药物是安全的。 本实验在我室原有抗hTNF-a表达载体的基础上,表达出可溶性hTNFa蛋白,之后用经典的细胞融合的方法获得抗hTNFa的杂交瘤细胞株；采用硫酸铵沉淀和蛋白G亲和层析法纯化腹水型单抗；采用ELISA酶链免疫分析方法测定其效价、抗体亚型和亲和力；采用MTT法测定抗体阻断hTNFa对L929细胞的细胞毒的作用和筛选拮抗型抗hTNFa单抗；流式细胞仪法测定抗体阻断hTNFa诱导L929细胞凋亡的作用。最终获得一株能稳定分泌抗hTNFa抗体的杂交瘤细胞株XB10,分泌的抗体亚型为IgG2；该抗体能高亲和力与hTNFa结合,特异性阻断hTNFa对L929细胞的细胞毒作用和抑制细胞凋亡的发生,并呈一定的剂量依赖性。用RT-PCR和PCR方法克隆出抗hTNFa的轻、重链基因,与T载体连接,测序结果与PDB库中抗体特征框架氨基酸序列相符合,是抗体的轻、重链基因。该研究为今后研制临床治疗型抗hTNFa的基因工程抗体奠定了实验基础。
[Abstract]:Tumor necrosis factor Tumor necrosis factor a (TNFa) is a multifunctional cytokine secreted by activated monocytes / macrophages. Studies have shown that appropriate amount of TNFa can help the host resist microbial invasion, kill some tumor cells or cause bleeding and necrosis of tumor tissue in vivo. However, excessive TNFa and other inflammatory factors together produce a variety of pathological damage, can cause tissue damage, severe septic shock. It can be said that TNF-a, as a key cytokine in regulating physiological balance, plays a "double-edged sword" role in human anti-tumor and other diseases. By inhibiting the activity of TNFa, it can be used to treat TNFa related diseases. At present, it is mainly used in rheumatoid arthritis and tumorigenesis. Growth and metastasis as well as treatment of multidrug resistance. A wide range of drugs have been developed targeting TNFa. These drugs inhibit the production and effect of TNFa from different levels and levels to achieve the purpose of anti-#en1# therapy. Clinical studies have shown that anti- TNFa drugs are safe. In this experiment, soluble hTNFa protein was expressed on the basis of the original anti-#en0# expression vector in our laboratory, and then the hybridoma cell line with anti-#en2# was obtained by classical cell fusion method. The ascites monoclonal antibody was purified by ammonium sulfate precipitation and protein G affinity chromatography. The titer, antibody subtype and affinity were determined by ELISA immunoassay. The cytotoxicity of hTNFa to L929 cells was blocked by MTT assay and the antagonistic anti hTNFa monoclonal antibody was screened. Flow cytometry was used to determine the blocking effect of hTNFa on apoptosis of L929 cells. Finally, a hybridoma cell line XB10, which could stably secrete anti-#en1# antibody, was obtained. The antibody subtype secreted was IgG2; The antibody can bind to hTNFa with high affinity and specifically block the cytotoxicity of hTNFa to L929 cells and inhibit the apoptosis of L929 cells. In a dose-dependent manner, the light and heavy chain genes of anti-#en2# were cloned by RT-PCR and PCR, and ligated with T vector. The results of sequencing are consistent with the amino acid sequence of the characteristic frame of antibody in PDB library and are light and heavy chain genes of antibody. This study will lay an experimental foundation for the development of clinical therapeutic antibody against hTNFa in the future.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】

相关期刊论文 前2条

1 侯勇;张奉春;黄烽;吴东海;鲍春德;倪立青;姚晨;;Infliximab治疗类风湿关节炎的随机双盲平行多中心临床试验[J];中华风湿病学杂志;2006年11期

2 王翔军;赵安成;;恶性肿瘤患者治疗前后血清一氧化氮及内皮素-1和血浆TNF-α及sTNF-R的检测及临床意义[J];实用医技杂志;2007年04期

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