抗人趋化素样因子1羧基端多肽抗体的制备、鉴定及组织芯片分析  

Preparation?熏 Identification?熏 and Analysis on Tissue Chips of Polyclonal Anti-peptide Antibody to Chemokine-like Factor 1

作　　者：石爽[1] 张颖妹[1] 邱晓彦[1] 朱晓辉[1] 刘雅楠[1] 甘启霓[1] 杨田[1] 宋泉声[1] 马大龙[1] 韩文玲[1] 

机构地区：[1]北京大学基础医学院免疫学系北京大学人类疾病基因研究中心,北京100083

出　　处：《中国医学科学院学报》2004年第5期496-499,i001,共5页Acta Academiae Medicinae Sinicae

基　　金：国家高技术研究发展计划项目(863项目)(2001AA215061;2002BA711A01);高等学校全国优秀博士论文作者专项资金(200257)资助~~

摘　　要：目的制备及鉴定抗人趋化素样因子1(CKLF1)多克隆抗体,为CKLF1表达和功能研究提供基础。方法利用生物信息学方法分析CKLF1蛋白序列,设计、合成CKLF1羧基端多肽,与钥孔戚血蓝素穴KLH雪偶联,免疫新西兰白兔获得多克隆抗体。经ELISA法测定抗体效价,Western-blot鉴定抗体与原核细胞体外翻译和果蝇S2细胞稳定转染表达的CKLF1反应,组织芯片检测该抗体与内源性表达的CKLFs结合。结果抗CKLF1C端多肽抗体的效价为10-4,能识别在原核细胞体外翻译系统和真核细胞中表达的CKLF1蛋白,也可与多种组织中天然表达的CKLFs发生特异性反应熏在正常支气管软骨、胃粘膜和平滑肌中呈阳性表达,在正常直肠和高分化直肠癌的腺上皮中呈强阳性表达,而在低分化的直肠癌中为阴性。结论抗CKLF1多克隆抗体效价高,亲和力强,可用于CKLF1的检测和研究。Objective To prepare the polyclonal anti-peptide antibody against chemokine-like factor1?穴CKLF1?雪and apply it to the expression and functional studies of CKLF1. Methods CKLF1 was analyzed with bioinformatics methods. The 16 amino acids sequence peptide was selected from CKLF1 C terminal end. Antibody was raised by immunizing rabbits with the peptide conjugated to keyhole limpet hemocyanin?穴KLH?雪. Results A high titer polycolonal antibody was obtained from the rabbit against the peptide. ELISA analysis proved that the titer of rabbit serum against anti-peptide of CKLF1 was up to 10-4. Western blot analysis revealed that it could react not only with recombinant CKLF1 expressed in a cell-Free Protein Biosynthesis System and Drosophila S2 cells?熏 but also recognize the endogenous CKLFs in the tissue array. Positive staining was detected in the normal bronchial cartilage?熏 gastric mucosa?熏 and gastric smooth muscle tissues. Normal rectum and well-differentiated rectal carcinoma showed strong positive staining?熏 but the poor-differentiated rectal carcinoma samples revealed negative staining. Conclusion The anti-peptide antibody can specifically recognize CKLFs and may be a useful reagent for the detection of CKLF1.

关 键 词：趋化素样因子1 抗多肽抗体 组织芯片 

分 类 号：R392.11[医药卫生—免疫学]