抗人T淋巴细胞CD4人鼠嵌合抗体真核表达的研究.pdf

CD4属于Ig超家族,为细胞膜表面单链糖蛋白,是HIV的主要受体。CD4+ T细胞参与大多数自身免疫性疾病的发生,而抗CD4的抗体可清除或抑制CD4+ T细胞,从而调节异常的T细胞应答。国外已对鼠源性抗CD4单抗用于治疗类风湿性关节炎、全身性红斑狼疮等自身免疫性疾病进行了广泛而深入的研究【1,2,3, 41,临床疗效良好。但是,鼠源性单抗应用于人体治疗将引起人抗鼠抗体(HAMA) 反应,反复使用可导致人体产生过敏反应且降低治疗效果,将鼠单抗改造为人. 鼠嵌合抗体能降低HAMA反应。嵌合抗体临床试用的大量研究表明,它临床疗效好,免疫原性与其它人源化抗体相似,而亲和力常常高于其它人源化抗体。本研究首先对质粒PAG4622+CD4VL和PAH4604+CD4VH进行双酶切及 DNA测序鉴定,证实其为含有鼠源性可变区和人源恒定区的嵌合抗体轻、重链表达载体。然后,绘制SP2/0细胞生长曲线确定SP2/0细胞最适生长条件,并通过抗生素筛选SP2/0细胞浓度的摸索,确定了组***醇、霉酚酸联合筛选对SP2/0 +/“。接着,采用电穿孔转染质粒DNA的方法,将制备好的线性质粒PAH4604+CD4VH和PAG4622+CD4VL共转染SP2/0 细胞,通过组***醇、霉酚酸联合筛选4~6周,获得了0925CASP2/0和 107CASP2/0两株阳性SP2/0细胞株。最后,通过ELISA、流式细胞术、RT-PCR 和DNA测序的方法对0925CASP2/0和1107CASP2/0细胞进行了初步鉴定。结果表明,本研究成功地在真核细胞SP2/,表达产量为1~2ng/ml。[关键词]: CD4嵌合抗体电穿孔SP2/0细胞真核表达 The Eukaryotic Expression ofAnti—human TLymphocyte CD4 Mouse/human Chimeric Antibody Ab stract As akind ofsingle chain glycoprotein on cellmembrane surface,CD4 belongs to Igsuper family and isalso the main receptor +T cellparticipates in majority autoimmune disease,and anti—CD4 antibody can purge orinhibitCD4+T cell, thereby it can beused to cure autoimmune prehensiVeness and in—depth researches towards mouse origin anti—CD4 monoclonal antibody designed to cure autoimmune disease(.,rheumatoid arthritis,systemic lupus erythematosus,et a1) hasbeen made[1,2,3,41,and curative amajor limitation tothe application ofmouse antibodies inhuman therapy isthehuman anti—mouseantibody (HAMA) using mouse antibodies may cause allergic reaction, and lower the therapeutic chimera antibody can reduce the HAMA response withtheoriginal antigen binding capability and low heterogeneity. Recent clinicalstudies showed that theantigenicity ofchimeric antibodies was similar tootherhumanized antibodies and theaffinity ofchimeric antibodies was beRer than otherhumanized antibodies. Endonuclease digesting and DNA sequencing confirmed th