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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 and identify polyclonal anti-human chemokine-like factor 1 (CKLF1) antibody and provide a basis for the study of the expression and function of CKLF1. METHODS: The CKLF1 protein sequence was analyzed by bioinformatics method. The CKLF1 carboxyl terminal polypeptide was designed and synthesized. The KLH-C conjugate was conjugated with keyhole limpet hemocyanin, and polyclonal antibody was immunized in New Zealand white rabbits. Antibody titers were determined by ELISA. The CKLF1 responses were analyzed by Western-blot in vitro and by stable transfection of Drosophila S2 cells. Tissue microarray was used to detect the binding of endogenous CKLFs. Results The titer of anti-CKLF1 C-terminal antibody was 10-4, which could recognize the CKLF1 protein expressed in the in vitro translation system and eukaryotic cells of prokaryotes. It could also react specifically with the naturally expressed CKLFs in various tissues. Bronchial cartilage, gastric mucosa and smooth muscle were positively expressed in the normal rectum and well-differentiated rectal adenocarcinoma was strongly positive expression in poorly differentiated rectal cancer was negative. Conclusion The anti-CKLF1 polyclonal antibody has high titer and high affinity and can be used for the detection and research of CKLF1.