兔抗人Rig-I蛋白N端CARD结构域多克隆抗体的制备与鉴定

Preparation and characterization of rabbit polyclonal antibody against the N terminal CARD domain of human Rig-I

目的:制备人维甲酸诱导基因I(hRig-I)N端CARD结构域(hRig-I-N)多克隆抗体。方法:从逆转录病毒载体MigRI-hRig-I中扩增hRig-I基因N端CARD结构域852bp长度的基因片段,克隆入pGEX-4T3中,转化大肠杆菌BL21(DE3),在IPTG诱导下,表达GST-hRig-I-N融合蛋白,将表达的融合蛋白纯化,免疫家兔制备抗体,并以间接ELISA法测定抗体效价,以Westernblot、细胞免疫荧光方法鉴定抗体的特异性。结果:在大肠杆菌中成功表达融合蛋白GST-hRig-I-N,ELISA法检测抗体的效价达到1∶125000,Western blot及细胞免疫荧光检测结果显示抗体可与原核及真核表达的hRig-I-N蛋白特异结合。结论:制备了高效价、高特异性的hRig-I-N抗体,为进一步研究hRig-I-N的功能奠定了基础。

AIM： To prepare the polyclonal antibody against the N-terminal CARD domain of human retinoic acidinduced gene I （bRig-I-N）. METHODS： The hRig-I gene of 852 bp in the N-terminal CARD domain was amplified from the constructed retrovirus plasmid MigRI-hRig-I and then cloned into the prokaryotic expression vector pGEX-4-1-3, the recombinant plasmid pGEX-4T3-hRig-I-N was transformed into E. coil BL21 （ DE3 ） to express fusion protein GST-hRig-I-N under the induction of IPTG, the fusion protein was purified and the antibody was made by the immunized rabbits, the titer of the polyclonal antibody was tested by the indirect ELISA, and specificity of the antibody was identified based on Western blot and cell immunofiuorescence. RESULTS. The fusion protein was successfully expressed, and titer of the antibody was up to 1 ： 125 000, by detection of Western blot and immunoflurescence it was indicated that the antibody combined well and specifically with expressed protein of hRig-t-N in prokaryotic and eukaryotic cells. CONCLUSION： The antibody of hRig-I-N with high titer and specificity was prepared, which lays a foundation for further studying the function of hRig-I-N.