Fusion Expression Of The Human First Extracellular Membrane Loop Chemokine Receptor 5 N-terminal And Preparation Of Its Specific Antibody F(ab')₂

Chemokines are small molecular superfamily that recruit leukocytes from the circulation to sites of infection.They fall into two major classes,based on the position of the first two cysteine residues:the CC and the CXC chemokines. Human P cheraokine receptor 5 is a specific receptor of CC subtribe chemokine receptor MIP-1 a,MIP-1 3 and RANTES. The CCR5 gene is located at 3q21.3,a CCR5 cDNA 1900 bp encoding 352 amino acids .CCR5 like the other chemokine receptors,is a seven- transmembrine domains protein,the seven- transmembrine domains is a helix and its amino acids have very high conservation. The extracellular 100pl(ECL1) and ECL2 was linked by disulfide bond to maintenanced the stability of the protein secondary structure.In recent years,we showed that CCR5 as a co-receptor could interact with HIV-1 infect ion.CCR5 was paid closed attention to since it was cloned in 1996.The aim is to- obtain the sequence of first extracillular domain of 3 chemokine receptor 5 (CCR5) N-terminal gene fragment with high level expression in E.coli and to prepare its specific antibodyF(ab');,and its detected method.Methods:We use computer analyzed and located the least homologous domain of the extracellular first 100p. The genefragment 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 NH2 -terminal antibody F(ab')2 was prpared by protein A-Sepharose CL-4B affinity chromatography,pepsin digestion and S-200 column preparation.Results:Sequencing was coincide with literature reports and by Reduced,unreduced SDS-PAGE and ELISA block examination analysis demonstrated that this F(ab')->had high specificity to combine wity huCCRS.Conclusion:In this paper,we show huCCR5 N-terminal gene fragment was cloned successfully and its specific antibody was prpared .By these we not only introduced a simple and quick method to get a specific antibody F(ab')2of certain functional domain but also a good idea and technique to study other high similar superfamily members.