抗人黑色素瘤嵌合抗体真核表达载体的构建与表达.pdfVIP

冯　蕾等　抗人黑色素瘤嵌合抗体真核表达载体的构建与表达　第 4 期 2·47 · 抗人黑色素瘤嵌合抗体真核表达载体的构建与表达① ( ) 冯 　蕾 　韩 　骅 　 第四军医大学基础部医学遗传与发育生物学教研室 ,西安 710033 　　中国图书分类号 　R39211 　　文献标识码 　A 　　文章编号 　1000484X( 2005) [摘 　要] 　目的 :构建嵌合型抗人黑色素瘤抗体的真核表达载体 ,并实现真核表达。方法 :从 3 株杂交瘤细胞中克隆得到 κ γ 鼠源单抗的可变区基因,插入含有抗人 Tac 抗原信号肽以及人免疫球蛋白 轻链恒定区基因和 1 重链恒定区基因的真核表 达载体 pMHCA 中 ,转染 293T 细胞 ,进行嵌合抗体表达 ,并用 RTPCR 、ELISA 、Western blot 免疫印迹等对抗体表达鉴定。结果 : 成功构建了抗人黑色素瘤人鼠嵌合抗体,并实现其真核表达。RTPCR 证实了该抗体在 mRNA 水平上的表达 ,ELISA 和 West ern blot 证实了抗人黑色素瘤人鼠嵌合抗体的表达。结论 :成功表达了抗人黑色素瘤人鼠嵌合抗体。 [ 关键词] 　嵌合抗体 ;黑色素瘤 ;基因工程 Construction and expression of mousehuman chimeric antihuman melanoma anti body FENG Lei , HAN Hua . Department of Medical Genetics and Developmental Bilogy , The Fourth Military Medical University , Xi ’an 710032 , China [ Abstract ] 　Objective :To construct eukaryotic expression vector for antihuman melanoma chimeric antibody ,and express the antibody in eukaryotic cells. Methods :Variable regions of the murine antibody were cloned from the antihuman melanoma hybridoma cell line HB8459 , HB8760 ,Mel3 ,and was inserted them into the chimeric antibody eukaryotic expression vector pMHCA. The constructed vector was transfected into 293T ,the chimeric antibody was expressed ,as detected by RTPCR ,ELISA ,and Western blot. Results :Eukaryotic expression vectors of the chimeric antibody against human melanoma were successfully constructed ,and expressed in eukaryotic cells. The expression of chimeric gene was confirmed by RTPCR ,specificity of the chimeric antibody was proved by ELISA and Western blot. Conclusion :Antihuman melanoma chi meric antibody was expressed successfully. [ Key words ] 　Chimeric antibody ;Melanoma ; Genetic engineering 　　黑色素瘤是一种恶性程度很高的肿瘤 ,早期即