抗RIP3抗体的制备及其亚细胞定位  

Preparation and subcellular localization of antibody against RIP3

作　　者：谢琳娜[1] 张娜[1] 陈明谅[1] 李勤喜[1] 周化民[1] 

机构地区：[1]厦门大学生命科学院,福建厦门361005

出　　处：《细胞与分子免疫学杂志》2006年第5期660-663,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金面上项目(30500273)

摘　　要：目的:制备兔抗受体相互作用蛋白3(RIP3)的抗体,并探讨其在鼠源NIH3T3细胞株中的定位。方法:用RTPCR技术从NIH3T3细胞株中克隆鼠rip3基因,进行原核表达及电洗脱纯化。以高纯度的RIP3抗原免疫新西兰兔制备抗血清并纯化。用Westernblot检测该抗体的特异性,以免疫荧光法确定RIP3在NIH3T3细胞株中的定位,以及TNFα和zVAD.fmk对其定位的影响。结果:获得特异性的兔抗RIP3蛋白的抗体。免疫荧光的结果显示,RIP3定位于细胞质中。单用TNFα或zVAD.fmk处理NIH3T3细胞后,RIP3的定位均无明显变化;而用zVAD.fmk和TNFα共同处理细胞后,在核内外均有RIP3分布。结论:成功地克隆rip3基因并制备高特异性的兔抗RIP3抗体。RIP3在NIH3T3细胞株中定位于细胞质中,为进一步研究RIP3在TNFα诱导的caspase非依赖的细胞凋亡途径中的作用奠定了基础。AIM： To determine the localization of RIP3 in NIH3T3 cell line and explore its roles in the signal transduction pathway of TNF-α-induced caspase-independent cell death. METHODS： RIP3 gene was cloned from NIH3T3 cell line by RT-PCR and the amino acid 287-486 sequence of RIP3 was overexpressed in E. coil strain BL2I, The protein was purified by electrodialysis to immune rabbits. Nine weeks later, the serum of the rabbits was harvested and anti-RIP3 polyclonal antibodies was purified and its specificity was detected by Western blot. Immunofluorescence was used to determine the subcellular localization of RIP3 in NIH3T3 cell line and the effects of TNF-α and z-VAD, fmk on RIP3 for its localization changing. RESULTS： A high concentration of rabbit polyclonal antibody against RIP3 with good immunological characteristics was obtained. RIP3 was successfully detected by Western blot with anti-RIP3 antibody in 293T cells transfected with pcDNA3-flag-RIP3 and in mouse NIH3T3 cell line. The results of immunoflue rescent staining displayed that RIP3 localized in cytoplasm in untreated NIH3T3 cells. Treatment with TNF-α or z-VAD, fmk alone didn＇t change its distribution. However in the cells induced by z-VAD, fmk and TNF-α, RIP3 was detected in both cytoplasm and nucleolus. CONCLUSION： The polyclonal antibody against RIP3 with high concentration and specificity was successfully obtained. In NIH3T3 cell line, RIP3 localized in cytoplasm, which was subject to change induced by z-VAD, fmk and TNF-α, suggesting that RIP3 plays an important role in TNF-α induced caspase-independent cell death signal transduction pathway.

关 键 词：RIP3 多克隆抗体 亚细胞定位 caspase非依赖的细胞凋亡途径 

分 类 号：R392.11[医药卫生—免疫学]