The preparation of first extracellular membrane loop1 of human monocyte chemotactic protein receptor 5 N- terminal's specific antibody F(ab′) 2 / 中国免疫学杂志

ABSTRACT

Objective:To prepare the first extracellular domain of human ? chemokine receptor 5(huCCR5)NH 2 terminal's specific antibody F(ab′) 2 and its detection method Methods:Use computer analyzed and located the least homologous domain of the extracellular first loops The gene fragment was amplified by PCR and cloned into the pGEX IN vector A recombinant GST fusion protein was constructed After comfirming the correctness of the inserted sequence,the transformation and expression of this fusion protein were performed in E coli The expression products of the fusion protein were purified and 2 New Zealand rabbits were immunized An anti huCCR5 NH 2 terminal antibody F(ab′) 2 was prepared by protein A Sepharose CL 4B affinity chromatography ,pepsin digestion and S 200 column seperation.Results:Reduced,unreduced SDS PAGE and ELISA block examination analysis demonstrated that this F(ab′) 2 had high specificity to combine with huCCR5 Conclusion:In this paper,not only introduce a simple and quick method to get a specific antibody F(ab′) 2 of certain functional domain but also a good idea and technique to study other high similar superfamily members