兔抗人CXCR3-B分子N端多肽多克隆抗体的制备与初步应用

Generation and preliminary application of rabbit anti-human polyclonal antibodies against NH_2-terminal peptides of CXCR3-B

目的： 获得兔抗人CXCR3-B分子N端第1～19、第17～35及第33～51个氨基酸多肽的多克隆抗体, 并对多克隆抗体进行初步鉴定和应用.方法： 应用Fmoc法化学合成CXCR3-B分子N端第1～19、第17～35及第33～51个氨基酸多肽, 经C18的RP-HPLC纯化后, 通过高碘酸钠法将纯化的CXCR3-B分子的多肽与KLH交联;皮下注射抗原免疫日本大耳白兔, 加强免疫得到抗血清, 应用蛋白G纯化获得多克隆抗体.对纯化的抗体进行ELISA、免疫印迹、免疫组化等初步鉴定和应用.结果： 分别化学合成CXCR3-B分子N端第1～19、第17～35及第33～51个氨基酸多肽, 纯化后多肽纯度分别为98%、 88.54%及80%, 达到免疫用抗原标准.多肽与KLH交联, 用于免疫动物.经纯化后的抗体效价分别为1∶ 32 000（0.1 mg/L）, 1∶ 4 000（3 mg/L）和1∶ 1 000（10 mg/L）.3种多肽的抗体可特异识别胎心组织中相对分子质量（Mr）约为50的CXCR3-B分子, 相应抗原定位于3种多肽的抗体可特异识别胎心组织中的血管内皮细胞.结论： 所制备的多克隆抗体可应用于ELISA、免疫印迹和免疫组化等实验, 为确定CXCR3-B分子的组织及细胞分布和定位、寻找CXCR3-B相互作用的分子提供了有力的工具.

AIM： To generate and identify the rabbit polyclonal antibodies against NH2-terminal peptides of CXCR3-B. METHODS： Three peptides （aa. 1 to 19, aa. 17 to 35, and aa. 33 to 51 ） of human CXCR3-B NH2-terminus were synthesized by using standard Fmoc. The synthesized peptides were purified by reversed phase high-performance liquid chromatography （RP-HPLC）, and cross-linked with keyhole limpet hemocyanin （KLH） by sodium metaperio- date. Rabbits were immunized with conjugated pepUdes for 3 times （400 μg/rabbit）. The polyclonal antibodies were pudried by Protein G from the collected antiserum. RESULTS： NH2-terminal peptides of human CXCR3-B with the purity of 98%, 88.54%, and 80%, respectively, were prepared. The titers of purified polyclonal antibodies were 1 ： 32 000 （0. 1 mg/L）, 1：4000 （3 mg/L）, and 1：1 000 （10 mg/L）, respectively. Western blot results showed that the antibodies could recognize the protein with molecular weight of .50 000 in the total lysates of human fetal heart, whereas the anti- bodies against the peptides of No. ! - 19 amino acids could also recognize an additional protein （40 000）, The antigens recognized by the antibodies were localized in the vascular endothelial cells of human fetal heart tissues, CONCLUSION： The polyclonal antibodies against NH2-terminal pep- tides generated and identified in the present work are specific to CXCR3-B protein and therefore can be useful tools for the functional study of human CXCR3-B.