兔抗人CD1d分子α3结构域抗体制备及鉴定.docVIP

兔抗人CD1d分子α3结构域抗体制备及鉴定 　 作者：陈章权, 黄震, 梁晓东, 何天文, 陆田田 【摘要】 　　目的: 制备兔抗人CD1d分子α3结构域（hCD1dα3）多克隆抗体。方法: PCR法扩增出人hCD1dα3编码区基因, 将其克隆入原核表达载体pET28中, 转化大肠杆菌BL21（DE3）, 用IPTG诱导重组蛋白的表达, 用Ni2+NTA Agarose亲和层析法纯化重组蛋白, 以之为免疫原免疫家兔, 制备多克隆抗体, 并用ELISA、 Western blot及免疫组织化学法检测抗体。结果: 成功构建了hCD1dα3原核表达载体pET28/hCD1dα3, 高效表达并纯化hCD1dα3蛋白, 间接ELISA检测所制备抗体的效价为1∶6400, Western blot显示该抗体能与hCD1d特异结合, 免疫组织化学法检测结果表明该抗体识别人小肠组织中的天然hCD1d。结论: 通过制备重组hCD1dα3蛋白为免疫原, 免疫家兔, 成功地制备了效价高、 特异性好的抗hCD1dα3多克隆抗体。 【关键词】 CD1d α3结构域 表达 抗体 制备 　　[Abstract] AIM: To prepare the rabbit antibody against the α3 domain of the human CD1d (hCD1dα3). METHODS: The gene fragment coding for hCD1dα3 was amplified by PCR and cloned into prokaryotic expression vector pET28, then expressed in E.coli BL21(DE3). The recombinant protein hCD1dα3 was purified with Ni2+NTA agarose column and used as immunogen to immunize the rabbit. The titer and specificity of the antihCD1dα3 antibody from the rabbit were analyzed by ELISA, Western blot and immunohistochemistry, respectively. RESULTS: The recombinant hCD1dα3 was successfully expressed and purified, and the polyclonal anithCD1dα3 antibody was successfully prepared. The titer of the antiserum was 1∶6400 by ELISA. Western blot analysis showed this antiboday reacted specifically with hCD1d. Immunohistochemistry analysis showed the antibody could recognize the native hCD1d in the human intestinal tissue. CONCLUSION: The antiCD1dα3 antibody from the rabbit with high titer and specificity has been prepared with purified recombinant hCD1dα3 as immunogen, which lays a foundation for further research into detection and functional study of CD1d. 　　[Keywords]CD1d; α3 domain; expression; antibody; preparation 　　CD1分子独立于MHC分子之外的一类特殊的抗原提呈分子。CD1d是其CD1家簇5个成员中的重要一员, 它提呈的抗原主要为脂质分子, 供NKT细胞识别[1], 在免疫调节、 NKT细胞的发育及与感染性疾病、 自身免疫性疾病、 肿瘤疾病的发生发展过程中起重要作用[2-4]。CD1d分子由胞外区、 跨膜区和胞内区组成, 其胞外区含3个结构域: 从氨基端开始依次为α1、 α2和α3结构域[5]。Kojo等[6]发现CD1d分子存在α3结构域缺失的变异体, 但尚未见进一步的生物学功能研究报道。我们在研究中也发现, 在一些肿瘤患者体内存在类似